Er:YAG Laser-Assisted Bone Regenerative Therapy for Implant Placement: A Case Series.

In order to achieve favorable ridge preservation (RP) or ridge augmentation (RA) in substantial vertical and/or horizontal bone defects and extraction sockets, a barrier membrane is usually employed. Recently, it was reported that a novel surgical technique for periodontal regenerative surgery applying Er:YAG laser (ErL) irradiation to form blood coagulation on the grafted bone surface, without using a membrane, resulted in sufficient bone regeneration in bone defects. This case series aims to present clinical and radiographic outcomes of ErL-assisted bone regenerative therapy (Er-LBRT), without use of membranes, for RP/RA before or after implant placement. In 10 cases, ErL irradiation was applied (50 mJ/pulse and 20 Hz without water spray in noncontact, defocused mode for approximately 60 seconds) to enhance the blood clot on the entire surface of the grafted bovine bone mineral before suturing. Wound healing was favorable without any postoperative complications such as wound gaping or infection of the grafted material. In all cases, dramatic bone regeneration was observed. After prosthetic treatment, peri-implant tissue and regenerated bone were stable and well-maintained during the follow-up period in each case. This novel technique of Er-LBRT without using a membrane resulted in favorable and stable RP/RA with sufficient bone regeneration for implant therapy.

Investigating the effect of antiseptic solution on the release of interleukin-6 and transforming growth factor beta 1 from human gingival fibroblasts using wound healing assays.

This in vitro study evaluated the effect of different antiseptics and different concentrations thereof in a model of wound healing using human gingival fibroblasts. The fibroblasts were rinsed with four different antiseptic solutions: sodium hypochlorite (HYP), hydrogen peroxide (H2O2), chlorhexidine digluconate (CHX), and benzalkonium chloride (BC). The effect on the release of interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF-ß1) was investigated using enzyme-linked immunosorbent assays (ELISAs). In addition, the effects of the antiseptics on wound healing at 1, 12, 24, and 48 h were assessed through a wound healing assay. The viability of the fibroblasts rinsed with antiseptics was investigated with respect to the concentrations inhibiting cell growth by 50% (IC50), 25% (IC25), and ≤2% (IC2). A statistically significant increased release of IL-6 was obtained with BC IC25 and IC2 after 12, 24, and 48 h (P < 0.01). For TGF-ß1, no significant release was found for CHX IC2 after 24 and 48 h or for IC50 and IC25 after 12 h. There was no significant effect on wound healing capacity for CHX or for BC IC25 and IC2. This study demonstrated that antiseptic rinses of human gingival fibroblasts alter the release of IL-6 and TGF-ß1 and impact wound healing capacity, with both BC and CHX conferring neutral effects.

Comparison of three block bone substitutes for bone regeneration: long-term observation in the beagle dog.

This study aimed to evaluate the impact of three types of block bone substitute material on bone formation and graft resorption in vivo. Standardized bone defects (n = 4 defects/animal) were created in the calvaria of nine dogs. Block bone substitutes made of deproteinized bovine bone mineral (DBBM), beta-tricalcium phosphate (ß-TCP) and a mixture alpha-TCP and hydroxyapatite (α-TCP/HA) were inserted into the bone defects. A fourth defect was left untreated (empty). All sites were covered with a collagenous membrane. Block biopsies were harvested at 3, 6 and 12 months post-implantation and analyzed by micro-CT and histology. Biomaterial absorption was minimal and incorporation within the defect margin was good for all biomaterials. However, ß-TCP demonstrated a relatively greater volume of new bone formation and less residual material volume when compared with DBBM and α-TCP/HA. Conversely, α-TCP/HA showed higher osteoconductive potential and a greater new bone area compared with the other two biomaterials. The block bone substitutes used in the present in vivo study showed advantageous in terms of maintenance of their original form in bony defect. However, the positive impact of all biomaterials on new bone formation and replacement of bone was minor even at 12 months. These findings indicate that block bone substitutes are not well suited to vertical bone augmentation. Further investigations are required to improve the insufficient new bone volume for promising clinical results.

Effect of Irrigation Time of Antiseptic Solutions on Bone Cell Viability and Growth Factor Release.

Antiseptic solutions are commonly utilized to treat local infection in the oral and maxillofacial region. However, surrounding vital bone is also exposed to antiseptic agents during irrigation and may have a potential negative impact on bone survival. The aim of the present study was therefore to investigate the effect of rinsing time with various antiseptic solutions on bone cell viability, as well as their subsequent release of growth factors important for bone regeneration. The bone samples collected from porcine mandible were rinsed in the following commonly utilized antiseptic solutions; povidone-iodine (0.5%), chlorhexidine digluconate (CHX, 0.2%), hydrogen peroxide (1%), and sodium hypochlorite (0.25%) for 1, 5, 10, 20, 30, or 60 minutes and assessed for cell viability and release of growth factors including vascular endothelial growth factor, transforming growth factor beta 1, bone morphogenetic protein 2, receptor activator of nuclear factor kappa-B ligand, and interleukin-1 beta by enzyme-linked immunosorbent assay. It was found in all the tested groups that the long exposure of any of the tested antiseptic solutions drastically promoted higher cell death. Sodium hypochlorite demonstrated the significantly highest cell death and at all time points. Interestingly, bone cell viability was highest in the CHX group post short-term rinsing of 1, 5, or 10 minutes when compared with the other 4 tested groups. A similar trend was also observed in subsequent growth factor release. The present study demonstrated that of the 4 tested antiseptic solutions, short-term CHX rinsing (ideally within 1 minute) favored bone cell viability and growth factor release. Clinical protocols should be adapted accordingly.

Periosteal distraction osteogenesis versus immediate periosteal elevation in a rat model: Histological and micro-CT analysis.

PURPOSE: The aim of the present study was to compare periosteal distraction osteogenesis (PDO) to immediate periosteal elevation (IPE) in terms of de novo bone formation. MATERIAL AND METHODS: Animals of PDO Group were subjected to a 7-day latency period and a 10-day distraction period. Distraction device in IPE Group were activated for 1 mm at placement. Both groups of animals were euthanized at 17, 31 and 45-day following surgery and the samples analyzed histologically and by micro-CT. Total gap region (TG) was divided in two subregions, less than 0.5 mm (LG) and over 0.5 mm of the gap height (HG). RESULTS: Bone formation in PDO Group was observed in the distal region of the distraction gap, whereas in IPE Group proximally and distally from the distraction gap. Bone volume increased in both groups in LG, HG and TG (p < 0.001), while bone mineral density only in HG (p = 0.001). More new bone was observed in PDO than in IPE Group in HG (p = 0.017) and in TG (p < 0.001), without differences found in bone mineral density. CONCLUSIONS: The function of immediately elevated periosteum is limited to the distance to the underlying bone. PDO may be successfully applied to maintain the osteogenic capacity of elevated periosteum.

Single-staged vs. two-staged implant placement in vertically deficient alveolar ridges using bone ring technique - Part 2: implant osseointegration.

OBJECTIVES: The aim of this study was to evaluate the osseointegration of implants placed in a single-staged compared to two-staged procedure using bone ring technique. MATERIAL AND METHODS: In this study were used standardized, vertical alveolar bone defects in dogs. In the test group, dental implants (Straumann BL® , Basel, Switzerland) were inserted simultaneously with bone ring technique. As control group served implants inserted 6 months following grafting. Implants of both groups were left for an osseointegration period of 3 and 6 months. The peri-implant bone loss and bone-to-implant contact within the bone ring and native bone were analyzed morphometrically. An explorative statistical analysis was performed. RESULTS: The peri-implant bone level remained relatively stable within groups and between groups per given time period. Most of bone apposite on the implant surface in two groups was composed of newly formed bone. A nonparametric analysis of variance (ANOVA) revealed no significant advantage for two-staged implant placement for new and total bone, except for residual bone (P = .0084). Furthermore, two groups of implants performed similarly in bone ring and in native bone throughout the observation period. CONCLUSIONS: In terms of osseointegration, both techniques are likely equally efficient in the present defect model. The single-staged implant placement with cortical bone grafts warrants further documentation in clinical studies.

Osteogenic potential of rhBMP9 combined with a bovine-derived natural bone mineral scaffold compared to rhBMP2.

OBJECTIVES: Combination therapies of growth factors and scaffolds for bone tissue engineering are becoming routine for clinical use. BMP9 has previously been characterized as one of the most osteogenic inducers among the BMP superfamily; however, up until recently, BMP9 has only been available through adenovirus transfection experiments (gene therapy). While recombinant human (rh)BMP2 is regarded as the gold standard for bone regeneration with recombinant growth factors, recently the successful development of rhBMP9 brings intriguing new possibilities for future clinical use. The purpose of this pioneering study was to investigate the effects of rhBMP9 in comparison with rhBMP2 on an in vitro cell behavior of bone-forming osteoblasts when combined with a bone grafting material. MATERIAL AND METHODS: Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto bovine-derived natural bone mineral (NBM) particles treated with (i) control, (ii) rhBMP2 (10 ng/ml), (iii) rhBMP2 (100 ng/ml), (iv) rhBMP9 (10 ng/ml) and (v) rhBMP9 (100 ng/ml). The effects of rhBMPs were compared for cell adhesion at 8 h, cell proliferation at 1, 3 and 5 days and osteoblast differentiation as assessed by real-time PCR at 3 and 14 days for genes encoding Runx2, collagen1alpha2 (COL1a2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, ALP staining and alizarin red staining were used to investigate localization of osteoblast differentiation marker and mineralization on NBM. RESULTS: Although neither rhBMP2 nor rhBMP9 influenced cell attachment to NBM particles, both were able to stimulate cell proliferation at 3 days. Furthermore, all concentrations of rhBMPs were able to significantly induce mRNA levels of Runx2, COL1a2 and OCN at 3 days. Interestingly, only rhBMP9 was able to significantly upregulate mRNA levels of ALP up to eightfold, and ALP staining up to 25-fold, when compared to rhBMP2. In addition, only rhBMP9 (100 ng/ml) significantly increased alizarin red staining when compared to control and rhBMP2 (10 ng/ml) samples. CONCLUSION: These results demonstrate that both rhBMP2 and rhBMP9 have osteopromotive properties on osteoblast differentiation. It was found that rhBMP9 additionally stimulated the osteopromotive potential of osteoblasts when compared to rhBMP2 by demonstrating higher levels of ALP expression and alizarin red staining. Further animal studies comparing both recombinant proteins are necessary to further characterize the osteoinductive potential of BMP9.

Effects of collagen membrane application and cortical bone perforation on de novo bone formation in periosteal distraction: an experimental study in a rabbit calvaria.

OBJECTIVES: The aim of the present study was to assess the impact of collagen membrane application and cortical bone perforations in periosteal distraction osteogenesis. STUDY DESIGN: A total of 32 New Zealand rabbits were randomized into four experimental groups, considering two treatment modalities. Calvarial bone was perforated or left intact (P+/-). In half the animals, the distraction mesh was covered with a collagen membrane (M+/-). All animals were subjected to a 7-day latency period and a 10-day distraction period. The samples were harvested after 4-week and 8-week consolidation periods and analyzed histologically and by means of micro-computed tomography. RESULTS: Primary, woven bone observed at the 4-week consolidation period was gradually replaced by lamellar bone at the 8-week consolidation period. Significant increase in bone volume was found in all groups (P < .001) and in bone mineral density in groups I (P-/M-; P < .001), III (P+/M-; P < .001), and IV (P+/M+; P = .013). Group III (P+/M-) showed significantly more new bone at the 8-week consolidation period compared with the other three groups (P = .001), with no differences observed in bone mineral density between groups at a given time-point. CONCLUSIONS: In the present model, cortical bone perforations have more impact on the osteogenic process compared with the application of a collagen membrane.

Collagen barrier membranes adsorb growth factors liberated from autogenous bone chips.

OBJECTIVE: Collagen membranes serve as barriers to separate bone grafts from soft tissues. Bone grafts harvested with a bone scraper release growth factors activating transforming growth factor-ß (TGF-ß) signaling in mesenchymal cells. The aim of the present pilot study was to determine whether collagen membranes adsorb molecules from bone-conditioned medium (BCM) with the capacity to provoke the expression of TGF-ß target genes in vitro. MATERIALS AND METHODS: Collagen membranes were soaked in aqueous extracts from fresh and demineralized bone chips placed in cell culture medium. Recombinant human TGF-ß1 served as control. Gingival fibroblasts were seeded onto the washed collagen membranes and evaluated for the expression of adrenomedullin, pentraxin 3, interleukin 11, and proteoglycan 4. Cell viability and morphology with phalloidin staining were also determined. RESULTS: Incubation of collagen membranes with BCM for at least one minute caused fibroblasts to decrease the expression of adrenomedullin and pentraxin 3, and to increase the expression of interleukin 11 and proteoglycan 4. Four different membrane treatments - incubated with recombinant TGF-ß1, pre-wetted with saline solution, exposed to UV light, and dry out and stored one week at room temperature - also provoked significant changes in gene expression. Likewise, conditioned medium from demineralized bone chips caused gene expression changes. BCM did not alter the viability or morphology of gingival fibroblasts. CONCLUSIONS: These findings demonstrate that collagen membranes rapidly adsorb the TGF-ß activity released from bone chips, a molecular process that might contribute to guided bone regeneration.

High-dose irradiation of bone chips preserves the in vitro activity of bone-conditioned medium.

Extracorporeal irradiation sterilizes resected tumor bone used as autografts in reconstruction surgery. Therapeutic irradiation is a standard technique in head and neck cancer therapy that aims to preserve organ function. Bone irradiation has a complex, mostly inhibitory, effect on remodeling and regeneration, although the underlying mechanisms are still not fully understood. It remains unclear if extracorporeal irradiation affects the paracrine-like activity of the corresponding autografts. We recently reported that bone-conditioned medium from autogenous bone chips contains a number of factors that might affect cell activity. In the present study, we investigated the effects of extracorporeal irradiation of porcine cortical bone chips on the activity of the corresponding bone-conditioned medium. The effects of bone-conditioned medium on the expressions of transforming growth factor-beta (TGF-ß) target genes in oral fibroblasts were assessed. Bone-conditioned medium from bone chips exposed to a total radiation dose up to 120 Gy did not affect expressions of TGF-ß target genes, including adrenomedullin, BTB/POZ domain-containing protein 11, proteoglycan 4, NADPH oxidase 4, and interleukin 11, in oral fibroblasts. In conclusion, bone irradiation does not alter the capability of the corresponding bone-conditioned medium to provoke a robust fibroblastic cell response in vitro. (J Oral Sci 58, 325-331, 2016).

In vitro effects of 0 to 120 Grays of irradiation on bone viability and release of growth factors.

BACKGROUND: High dose radiation therapy is commonly used in maxillofacial surgeries to treat a number of head and neck tumors. Despite its widespread use, little information is available regarding the effects of irradiation on bone cell viability and release of growth factors following dose-dependent irradiation. METHODS: Bone samples were collected from porcine mandibular cortical bone and irradiated at doses of 0, 7.5, 15, 30, 60 and 120 Grays. Thereafter, cell viability was quantified, and the release of growth factors including TGFß1, BMP2, VEGF, IL1ß and RANKL were investigated over time. RESULTS: It was observed that at only 7.5Gy of irradiation, over 85 % of cells were non-vital and by 60 Gy, all cells underwent apoptosis. Furthermore, over a 7-fold decrease in VEGF and a 2-fold decrease in TGFß1 were observed following irradiation at all tested doses. Little change was observed for BMP2 and IL1ß whereas RANKL was significantly increased for all irradiated samples. CONCLUSIONS: These results demonstrate the pronounced effects of irradiation on bone-cell vitality and subsequent release of growth factors. Interestingly, the largest observed change in gene expression was the 7-fold decrease in VEGF protein following irradiation. Future research aimed at improving our understanding of bone following irradiation is necessary to further improve future clinical treatments.

Single-staged vs. two-staged implant placement using bone ring technique in vertically deficient alveolar ridges - Part 1: histomorphometric and micro-CT analysis.

BACKGROUND: Simultaneous implant placement with bone grafting shortens the overall treatment period, but might lead to the peri-implant bone loss or even implant failure. The aim of this study was to compare the single-staged to two-staged implant placement using the bone ring technique. MATERIAL AND METHODS: Four standardized alveolar bone defects were made in the mandibles of nine dogs. Dental implants (Straumann BL® , Basel, Switzerland) were inserted simultaneously with bone ring technique in test group and after 6 months of healing period in control group. Animals of both groups were euthanized at 3 and 6 months of osseointegration period. The harvested samples were analyzed by means of histology and micro-CT. RESULTS: The amount of residual bone decreased while the amount of new bone increased up to 9 months of healing period. All morphometric parameters remained stable between 3 and 6 months of osseointegration period within groups. Per a given time point, median area of residual bone graft was higher in test group and area of new bone in control group. The volume of bone ring was greater in test than in control group, reaching the significance at 6 months of osseointegration period (P = 0.002). CONCLUSIONS: In the present type of bone defect, single-staged implant placement may be potentially useful to shorten an overall treatment period.

Recombinant Human Bone Morphogenetic Protein 9 (rhBMP9) Induced Osteoblastic Behavior on a Collagen Membrane Compared With rhBMP2.

BACKGROUND: Bone morphogenetic protein 9 (BMP9) has previously been characterized as one of the most osteogenic growth factors of the BMP family. To the best of the authors' knowledge, previous experiments have only used adenovirus transfection (gene therapy). With the recent development of recombinant human BMP9 (rhBMP9), the present study investigates the osteopromotive potential of BMP9 versus rhBMP2 when loaded onto collagen membranes. METHODS: ST2 stromal bone marrow cells were seeded onto: 1) control; 2) low-dose rhBMP2 (10 ng/mL); 3) high-dose rhBMP2 (100 ng/mL); 4) low-dose rhBMP9 (10 ng/mL); and 5) high-dose rhBMP9 (100 ng/mL) porcine collagen membranes. The following parameters were compared among groups: 1) cell adhesion (at 8 hours); 2) cell proliferation (at 1, 3, and 5 days); 3) real-time polymerase chain reaction for genes encoding runt-related transcription factor 2; 4) alkaline phosphatase (ALP); 5) bone sialoprotein ([BSP] at 3 and 14 days); and 6) alizarin red staining (at 14 days). RESULTS: rhBMP2 and rhBMP9 demonstrated little effect on cell attachment and proliferation; however, pronounced increases were observed in osteoblast differentiation. All groups significantly induced ALP messenger RNA (mRNA) levels at 3 days and BSP levels at 14 days; however, high-dose rhBMP9 showed significantly higher values compared with all other groups for ALP levels (five-fold increase at 3 days and two-fold increase at 14 days). Alizarin red staining further revealed both concentrations of rhBMP9 induced up to three-fold more staining compared with rhBMP2. CONCLUSIONS: Results indicate that the combination of collagen membranes with rhBMP9 induced significantly higher ALP mRNA expression and alizarin red staining compared with rhBMP2. These findings suggest that rhBMP9 may be a suitable growth factor for future regenerative procedures in bone biology.

Comparative release of growth factors from PRP, PRF, and advanced-PRF.

OBJECTIVES: The use of platelet concentrates has gained increasing awareness in recent years for regenerative procedures in modern dentistry. The aim of the present study was to compare growth factor release over time from platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and a modernized protocol for PRF, advanced-PRF (A-PRF). MATERIALS AND METHODS: Eighteen blood samples were collected from six donors (3 samples each for PRP, PRF, and A-PRF). Following preparation, samples were incubated in a plate shaker and assessed for growth factor release at 15 min, 60 min, 8 h, 1 day, 3 days, and 10 days. Thereafter, growth factor release of PDGF-AA, PDGF-AB, PDGF-BB, TGFB1, VEGF, EGF, and IGF was quantified using ELISA. RESULTS: The highest reported growth factor released from platelet concentrates was PDGF-AA followed by PDGF-BB, TGFB1, VEGF, and PDGF-AB. In general, following 15-60 min incubation, PRP released significantly higher growth factors when compared to PRF and A-PRF. At later time points up to 10 days, it was routinely found that A-PRF released the highest total growth factors. Furthermore, A-PRF released significantly higher total protein accumulated over a 10-day period when compared to PRP or PRF. CONCLUSION: The results from the present study indicate that the various platelet concentrates have quite different release kinetics. The advantage of PRP is the release of significantly higher proteins at earlier time points whereas PRF displayed a continual and steady release of growth factors over a 10-day period. Furthermore, in general, it was observed that the new formulation of PRF (A-PRF) released significantly higher total quantities of growth factors when compared to traditional PRF. CLINICAL RELEVANCE: Based on these findings, PRP can be recommended for fast delivery of growth factors whereas A-PRF is better-suited for long-term release.

Effects of Antiseptic Solutions Commonly Used in Dentistry on Bone Viability, Bone Morphology, and Release of Growth Factors.

PURPOSE: Antiseptic solutions are commonly used in dentistry for a number of sterilization procedures, including harvesting of bone chips, irrigation of extraction sockets, and sterilization of osteonecrotic bone. Despite its widespread use, little information is available regarding the effects of various antiseptic solutions on bone cell viability, morphology, and the release of growth factors. MATERIALS AND METHODS: The antiseptic solutions included 1) 0.5% povidone iodine (PI), 2) 0.2% chlorhexidine diguluconate (CHX), 3) 1% hydrogen peroxide (H2O2), and 4) 0.25% sodium hypochlorite (HYP). Bone samples collected from porcine mandibular cortical bone were rinsed in the antiseptic solutions for 10 minutes and assessed for cell viability using an MTS assay and protein release of transforming growth factor (TGF-ß1), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor (VEGF), interleukin (IL)-1ß, and receptor activator of nuclear factor κB ligand (RANKL) using an enzyme-linked immunosorbent assay at 15 minutes and 4 hours after rinsing. RESULTS: After antiseptic rinsing, changes to the surface protein content showed marked alterations, with an abundant protein layer remaining on CHX-rinsed bone samples. The amount of surface protein content gradually decreased in the following order: CHX, H2O2, PI, and HYP. A similar trend was also observed for the relative cell viability from within bone samples after rinsing, with up to 6 times more viable cells found in the CHX-rinsed bone samples than in the HYP- and PI-rinsed samples. An analysis of the growth factors found that both HYP and PI had significantly lower VEGF and TGF-ß1 protein release from bone samples at 15 minutes and 4 hours after rinsing compared with CHX and H2O2. A similar trend was observed for RANKL and IL-1ß protein release, although no change was observed for BMP2. CONCLUSIONS: The results from the present study have demonstrated that antiseptic solutions present with very different effects on bone samples after 10 minutes of rinsing. Rinsing with CHX maintained significantly higher cell viability and protein release of growth factors potent to the bone remodeling cycle.

Antiseptic solutions modulate the paracrine-like activity of bone chips: differential impact of chlorhexidine and sodium hypochlorite.

AIM: Chemical decontamination increases the availability of bone grafts; however, it remains unclear whether antiseptic processing changes the biological activity of bone. MATERIALS AND METHODS: Bone chips were incubated with four different antiseptic solutions including (1) povidone-iodine (0.5%), (2) chlorhexidine diguluconate (0.2%), (3) hydrogen peroxide (1%) and (4) sodium hypochlorite (0.25%). After 10 min. of incubation, changes in the capacity of the bone-conditioned medium (BCM) to modulate gene expression of gingival fibroblasts was investigated. RESULTS: Conditioned medium obtained from freshly prepared bone chips increased the expression of TGF-ß target genes interleukin 11 (IL11), proteoglycan4 (PRG4), NADPH oxidase 4 (NOX4), and decreased the expression of adrenomedullin (ADM), and pentraxin 3 (PTX3) in gingival fibroblasts. Incubation of bone chips with 0.2% chlorhexidine, followed by vigorously washing resulted in a BCM with even higher expression of IL11, PRG4 and NOX4. These findings were also detected with a decrease in cell viability and an activation of apoptosis signalling. Chlorhexidine alone, at low concentrations, increased IL11, PRG4 and NOX4 expression, independent of the TGF-ß receptor I kinase activity. In contrast, 0.25% sodium hypochlorite almost entirely abolished the activity of BCM, whereas the other two antiseptic solutions, 1% hydrogen peroxide and 0.5% povidone-iodine, had relatively no impact respectively. CONCLUSION: These in vitro findings demonstrate that incubation of bone chips with chlorhexidine differentially affects the activity of the respective BCM compared to the other antiseptic solutions. The data further suggest that the main effects are caused by chlorhexidine remaining in the BCM after repeated washing of the bone chips.

Bone Conditioned Medium: Preparation and Bioassay.

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-ß regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

Conditioned medium of demineralized freeze-dried bone activates gene expression in periodontal fibroblasts in vitro.

BACKGROUND: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-ß signaling in mesenchymal cells. The aim of this study is to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid, and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium obtained from DBM (DBCM). Changes in the expression of TGF-ß target genes were determined. RESULTS: DBCM altered the expression of TGF-ß target genes (e.g., adrenomedullin, pentraxin 3, KN motif and ankyrin repeat domains 4, interleukin 11, NADPH oxidase 4, and BTB [POZ] domain containing 11) by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-ß receptor type I kinase inhibitor SB431542 [4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide] but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-ß target genes. CONCLUSION: The findings suggest that the DBCM can activate TGF-ß signaling in oral fibroblasts.

Evaluation of cortical bone thickness and root proximity at maxillary interradicular sites for mini-implant placement.

OBJECTIVES: The purpose of this study was to evaluate the interradicular cortical bone thickness, alveolar process width and root proximity for planning mini-implant placement in the maxillary alveolar process. MATERIAL AND METHODS: Eighty maxillae (right and left sides) of 40 Japanese adult skulls were examined. The samples were imaged and measured using a micro-CT system. Buccal and palatal interradicular cortical bone thickness, alveolar process width, and root proximity were measured in six interradicular sites from distal of central incisor to mesial of second molar. Buccal and palatal interradicular cortical bone thickness and alveolar process width were measured at 10 different vertical levels. Root proximity was measured at four different vertical levels. RESULTS: Buccal and palatal interradicular cortical bone thickness and alveolar process width tended to increase from crest to base of alveolar process. The buccal interradicular cortical bone thickness between canine and first premolar or between first premolar and second premolar was the greatest, and between central incisor and lateral incisor was the least. The palatal interradicular cortical bone was significantly thicker than the buccal. The root proximity between second premolar and first molar or first premolar and second premolar was the widest and between central incisor and lateral incisor it was the narrowest. CONCLUSIONS: The findings of this study suggest that recommendations when low dose 3D multislice CT or low dose cone beam imaging is not available, the results of this research may be useful in providing indicators for selecting the design of the placement site.

25-gauge vitrectomy versus intravitreal bevacizumab for macular edema secondary to branch retinal vein occlusion: 1 year follow-up.

INTRODUCTION: This study aims to compare the long-term efficacy of 25-gauge vitrectomy to that of intravitreal bevacizumab (IVB) for the treatment of macular edema (ME) secondary to branch retinal vein occlusion (BRVO). MATERIALS AND METHODS: The medical records of 46 eyes of 46 consecutive patients were reviewed. Twenty-seven eyes underwent 25-gauge vitrectomy (VIT Group) and 19 eyes received 1.25 mg of IVB (IVB Group). The best-corrected visual acuities (BCVAs) in logarithm of minimum angle resolution units and central macular thicknesses (CMTs) were evaluated before and 3, 6, and 12 months after the initial treatment. RESULTS: There was no significant difference in the pre-treatment BCVA and CMT between the 2 groups. In the VIT Group, the preoperative BCVA was 0.59 and the CMT was 587.3 µm and the BCVA was 0.35 and the CMT was 286.6 µm, 12 months after the vitrectomy. Both values were significantly (P <0.05) better at 12 months than the preoperative values. In the IVB Group, the average number of IVB was 2.4 during the 1-year period. The BCVA was 0.69 and the CMT was 590.9 µm before the IVB, and the BCVA was 0.36 and the CMT was 360.1 µm, 12 months after the initial IVB. The improvements of these 2 parameters were significant (P <0.05) at 12 months after the initial IVB. The differences in the BCVA and CMT at 12 months between the 2 groups were not significant. CONCLUSION: These results suggest that the 25-gauge vitrectomy and IVB have similar effects in improving the BCVA and CMT in eyes with ME secondary to BRVO. However, IVB often required several injections to preserve the improvement.