Osteogenic and long-term antibacterial properties of Sr/Ag-containing TiO2 microporous coating in vitro and in vivo.

Bacterial infection and poor osseointegration are two critical issues that need to be solved for long-term use of titanium implants. As such, Sr/Ag-containing TiO2 microporous coatings were prepared on a Ti alloy surface in the current study via a single-step microarc oxidation technique. The coatings showed both good cytocompatibility in vitro and biosafety in vivo. Sr/Ag incorporation brought no significant change in the surface micromorphology and physicochemical properties, but endowed the coating with strong osteogenic activity and long-term antibacterial capability in vitro. Furthermore, the osteogenic and antibacterial capability of the coating was also confirmed in vivo. In a rat osseointegration model, new bone formation, implant-bone contact, removal torque and bone mineralization were all significantly increased in the M-Sr/Ag group when compared with those in group M, although they were slightly lower than those in group M-Sr. In a periimplantitis model, no rats suffered infection in the M-Sr/Ag group after 3 months of osseointegration and 5 weeks of bacterial inoculation period, when compared to 100% and 75% infection rates in M and M-Sr groups, respectively. In addition, active bone remodeling and many mesenchymal cells were observed in the M-Sr group, suggesting good bone regeneration potential in Sr-containing coatings in the case of controlled periimplantitis. Overall, the Sr/Ag-containing TiO2 microporous coating is valuable for preventing periimplantitis and improving implant reosseointegration, and is therefore promising for long-term and high quality use of titanium implants.

The effect of strontium content on physicochemical and osteogenic property of Sr/Ag-containing TiO2 microporous coatings.

Strontium (Sr) is the most common element introduced into TiO2 coatings to strengthen the osteogenic property of titanium implants. However, the optimal Sr content and its effect on osteogenic and physicochemical properties of the coatings need to be clarified. In the current study, TiO2 microporous coatings with different contents of Sr (9.64-21.25 wt %) and silver (Ag) (0.38-0.75 wt %) were prepared via micro-arc oxidation technique. Sr contents did not change physicochemical properties of the coatings, including surface microstructure, micropore size and distribution, phase composition, roughness and hydrophilicity. Meanwhile, higher Sr contents (18.23-21.25 wt %) improved cytocompatibility, proliferation and alkaline phosphatase (ALP) activity of preosteoblasts, even the coatings underwent 30 days' PBS immersion. Furthermore, higher Sr contents facilitated preosteoblast growth and spreading, which are essential for their proliferation and osteogenic differentiation. Therefore, it is promising to incorporate higher Sr content (18.23-21.25 wt %) within TiO2 microporous coatings to improve their osteogenic capability.

Classification and research progress of implant surface antimicrobial techniques.

Due to the good biocompatibility and ideal mechanical property, titanium implants have been widely used in dental clinic and orthopedic surgery. However, bacteria induced infection can cause per-implant inflammation and decrease the success rate of implant surgery. Therefore, developing antimicrobial techniques is essential to successful application of titanium implants. Many surface antimicrobial techniques, including antimicrobial coating and surface modifications, have been explored and they always exert antimicrobial effect by reducing bacterial adhesion, inhibiting their metabolism, or destructing cell structure. In this paper, different surface antimicrobial techniques and their recent research progress are reviewed to provide a brief insight on this area.

Osteogenic capability of strontium and icariin-loaded TiO2 nanotube coatings in vitro and in osteoporotic rats.

Titanium (Ti) and Ti alloys are widely used biomaterials, but they lack osteogenic capability for rapid bone integration. To improve osseointegration of Ti implants, TiO2 nanotubes were prepared using the anodizing oxidation technique, and strontium (Sr) combined with icariin (ICA) was loaded on TiO2 nanotube coatings. Cell adhesion and proliferation of MC3T3-E1 cells, alkaline phosphatase (ALP) activity, mineralization of extracellular matrix, and bone formation around titanium implants in ovariectomized rats, were examined separately. The results showed that compared with pure Ti, TiO2 and Sr-loaded TiO2 coatings, the coatings loaded with both Sr and ICA showed better effect on cell adhesion and proliferation, higher ALP activity and more red-stained mineralized nodules. Furthermore, more bone was formed around implants loaded with both Sr and ICA in osteoporotic rats. Therefore, coating with Sr and ICA is valuable for clinical application to strengthen the osseointegration of titanium implants, especially in osteoporotic patients.

Evaluation of osteogenic and antibacterial properties of strontium/silver-containing porous TiO2 coatings prepared by micro-arc oxidation.

Ti and Ti alloys are bioinert materials and two frequent problems associated with them are bacterial infection and lack of osteogenic potential for rapid bone integration. To overcome the problems, the present study incorporated strontium (Sr) and silver (Ag) simultaneously into porous TiO2 coatings through a single-step technique, micro-arc oxidation (MAO). Incorporation of Sr and Ag brought no significant changes to coating micromorphology and physicochemical properties, but endowed TiO2 coatings with both strong antibacterial activity and osteogenic ability. Antibacterial activity increased with Ag contents in the coatings. When Ag content reached 0.58 wt%, the coating showed both excellent short-term (100.0%) and long-term (77.6%) antibacterial activities. Sr/Ag-containing coatings with 18.23 wt% Sr and 0.58 wt% Ag also presented good cytocompatibility for preosteoblast adhesion and proliferation, and promoted preosteoblast osteogenic differentiation both short-termly and long-termly. However, higher Ag content (1.29 wt%) showed toxic effects to preosteoblasts. In summary, MAO is a simple and effective way to incorporate Sr and Ag into porous TiO2 coatings and Sr/Ag-containing TiO2 coating with 18.5 wt% Sr and 0.58 wt% Ag has both good osteogenic activity and strong antibacterial capability short-termly and long-termly. Therefore, such coatings are valuable for clinical application to strengthen osseointegration and long-term high quality use of titanum implants.

Effectiveness of strontium-doped brushite, bovine-derived hydroxyapatite and synthetic hydroxyapatite in rabbit sinus augmentation with simultaneous implant installation.

Various bone substitutes have been applied in sinus augmentation (SA) to overcome insufficient bone height at the posterior maxilla region caused by pneumatized sinus and severe alveolar bone resorption after teeth loss. However, their effectiveness in SA needs to be further elucidated. In this study, strontium-doped brushite (Sr-DCPD), a new bone substitute, together with bovine-derived hydroxyapatite (bHA) and synthetic hydroxyapatite (sHA) was used in rabbit maxillary SA with simultaneous implant installation. The sinus space-keeping capacity, resorption rate, osteoconductivity, and mechanical properties of regenerated bone, were evaluated by micro-computed tomography (CT), histological analysis, and mechanical testing. Sr-DCPD exhibited the best osteoconductivity and new bone formation (<4 weeks), but its final bone regeneration and removal torque of implants at week 12 were the lowest, mainly due to its poor space-keeping capacity and fast resorption. bHA exhibited the best space-keeping capacity and slowest resorption rate, but relative lower final bone volume and mechanical properties, while sHA showed good space-keeping capacity, slower resorption rate, and the best final bone formation and mechanical properties. sHA was most effective for SA and bHA was also an acceptable bone substitute; however, Sr-DCPD was least effective and not suitable in SA by itself.

CaMKII(δ) regulates osteoclastogenesis through ERK, JNK, and p38 MAPKs and CREB signalling pathway.

Calcium/calmodulin-dependent protein kinases (CaMKs) are a group of important molecules mediating calcium signal transmission and have been proved to participate in osteoclastogenesis regulation. CaMKII, a subtype of CaMKs is expressed during osteoclast differentiation, but its role in osteoclastogenesis regulation remains controversial. In the present study, we identified that both mRNA and protein levels of CaMKII (δ) were upregulated in a time-dependent manner during osteoclast differentiation. CaMKII (δ) gene silencing significantly inhibited osteoclast formation, bone resorption, and expression of osteoclast-related genes, including nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), and c-Src. Furthermore, CaMKII (δ) gene silencing downregulated phosphorylation of mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38, which were transiently activated by RANKL. Specific inhibitors of ERK, JNK, and p38 also markedly inhibited expression of osteoclast-related genes, osteoclast formation, and bone resorption like CaMKII (δ) gene silencing. Additionally, CaMKII (δ) gene silencing also suppressed RANKL-triggered CREB phosphorylation. Collectively, these data demonstrate the important role of CaMKII (δ) in osteoclastogenesis regulation through JNK, ERK, and p38 MAPKs and CREB pathway.

[Multiple pulp stones: report of a case and literature review].

Pulp stones were denaturation of pulp tissue, which were usually found in the pulp chamber. Generally, they were associated with caries and pulposis, and the occurrence of pulp stone increased with age. Pulp stones were frequently found by radiographic examination, and appeared as radiopaque lesions which were round or ovoid in shape. We reported an unusual case of multiple pulp stones with normal clinical crowns in a young female patient and analyzed the possible etiology.

Low-magnitude high-frequency loading, by whole-body vibration, accelerates early implant osseointegration in ovariectomized rats.

Osteoporosis deteriorates jaw bone quality and may compromise early implant osseointegration and early implant loading. The influence of low­magnitude, high­frequency (LMHF) vibration on peri­implant bone healing and implant integration in osteoporotic bones remains poorly understood. LMHF loading via whole­body vibration (WBV) for 8 weeks has previously been demonstrated to significantly enhance bone­to­implant contact, peri­implant bone fraction and implant mechanical properties in osteoporotic rats. In the present study, LMHF loading by WBV was performed in osteoporotic rats, with a loading duration of 4 weeks during the early stages of bone healing. The results indicated that 4­week LMHF loading by WBV partly reversed the negative effects of osteoporosis and accelerated early peri­implant osseointegration in ovariectomized rats.

[Literature analysis of current bilingual teaching on stomatology in China].

PURPOSE: To investigate the current status and problems of bilingual teaching on stomatology in China. METHODS: Three database, including CNKI, Wanfang database and VIP were used to retrieve the articles of bilingual teaching on stomatology from January 2001 to December 2012. RESULTS: Fifty articles were included in this study and the following items, publishing time, foundation support, journals, authors and author affiliation, the course, teaching effects, research classification and main components of the articles were analyzed. The problems remained were also discussed. CONCLUSIONS: The level of current bilingual teaching on stomatology in China is low and great efforts are needed to make it better. Supported by Research Fund of Hebei United University(Z201333).

Early healing of hydroxyapatite-coated implants in grafted bone of zoledronic acid-treated osteoporotic rabbits.

BACKGROUND: Resorption of grafted bone and delayed osseointegration of implants are main problems associated with alveolar bone augmentation in dental implantology, especially for patients with osteoporosis. The aim of this study is to investigate the early healing response of implants to systemic treatment of zoledronic acid (ZA) in autogenous grafted iliac bone of osteoporotic rabbits. METHODS: Ovariectomy (OVX) or sham operation was performed in 46 rabbits, and osteoporotic changes were verified in animals receiving OVX 3 months later. The remaining animals were divided into three groups (n = 12): sham, OVX, and OVX with ZA treatment (ZA group). Autogenous iliac bone grafting was performed in bilateral tibiae, and hydroxyapatite-coated titanium implants were simultaneously placed into the grafted bone. The animals were sacrificed 2 and 8 weeks later for examination. RESULTS: At both time points, systemic treatment of ZA efficiently promoted bone healing of implants in grafted bone, and all histologic and microcomputed tomography bone indices, including mineralized bone volume, implant-bone contact ratio, connectivity density, trabecular thickness, and trabecular number, were significantly increased in the ZA group compared with the OVX-only group (P <0.01); implant-bone contact rates in the ZA group were even restored to levels similar to those of sham-operated animals (P >0.05). Furthermore, biomechanical testing demonstrated that removal torque of implants was significantly increased in the ZA group compared with the OVX group (P <0.01). CONCLUSION: Systemic treatment with ZA could efficiently promote early bone healing of implants in autogenous grafted bone of osteoporotic rabbits by increasing early osseointegration and fixation of implants.

[Influence of zoledronate on osteoclast differentiation and gene expression of calmodulin and calmodulin-dependent protein kinase II].

OBJECTIVE: To investigate the effect of zoledronate acid on osteoclast differentiation and gene expression of calmodulin (CAM) and calmodulin-dependent protein kinase (CAMK)II. METHODS: Receptor activation of nuclear factor κB ligand (RANKL) was used to induce differentiation of RAW264.7 cells into osteoclasts in vitro. The cells were divided into two groups, group A and group B. Both groups were treated with RANKL for 5 days, whereas group B was also treated with zoledronate for the last 2 days.Osteoclastogenesis and gene expression of CAM and CAMK II were examined. RESULTS: In group B, the number of new-generated osteoclasts (≥3 nuclei), number and size of dentin resorption lacunaes were (23 ± 3) , (19 ± 2) and (4951 ± 223) µm(2) respectively, which were significantly lower than those [(44 ± 3) , (46 ± 1) and (13 331 ± 248) µm(2)] in group A (P < 0.01).mRNA and protein level of CAM and CAMK II were also significantly down-regulated in group B when compared with group A (P < 0.01) and the decrease was 26.7% and 37.2% respectively for CAM, 57.0% and 76.1% respectively for CAMK II. CONCLUSIONS: Zoledronate acid could significantly inhibit formation and resorption function of osteoclasts. CAM and CAMKII may be involved in the inhibition process.

[Effect of glial cell derived neurotrophic factor on regeneration of facial nerve defects by autogenous vein conduit].

OBJECTIVE: To study the effects of glial cell derived neurotrophic factor (GDNF) on regeneration of facial nerve defects by autogenous facial vein conduit. METHODS: Thirty-six rabbits were used in this study and 10 mm-length facial nerve defects were made on both sides of all animals. The nerve gaps were bridged using autogenous posterior facial vein graft of the same side. The animals received injection of either saline (group A, n=16) or GDNF (group B, n=16) into the veins. Nerve function was evaluated by evoking nerve action potential immediately after operation and 4, 8 and 16 weeks after operation. Regenerated nerve samples were harvested at 4, 8, and 16 weeks after operation and processed for histology and transmitting electron microscopic examination (TEM). RESULTS: Action potential did not exist immediately after operation but it was evoked at 4, 8, and 16 weeks in both groups. At 4 and 8 weeks after operation, the amplitude and width of action potential were significantly higher in group B than group A (P < 0.01), except wave width at 4 weeks, which showed no significant differences, while the latency period was significantly shorter in group B than that in group A (P < 0.01). At 16 weeks, action potential was similar between two groups, except wave amptitude, which was higher in group B than group A (P < 0.01). Morphologic and TEM examinations showed more matured myelinated nerve fibers and active Schwann's cells in group B when compared group A during the whole regeneration process. CONCLUSION: GDNF can promote nerve regeneraat early stage during reconstruction of facial nerve defects by autogenous facial vein conduit and combination of GDNF and autogenous vein graft provides a valuable method for clinical reconstruction of facial nerve defects.

[The effect of experimental osteoporosis on bone healing of autologous iliac crest graft around implants].

OBJECTIVE: To investigate the influences of experimental osteoporosis (OP) on bone healing of autologous iliac crest graft around dental implants in rabbits. METHODS: Twenty Japanese rabbits were randomly divided into two groups. Bilaterally ovariectomy was performed on experimental group and control group received sham-operation. Twelve weeks later, femoral bones were examined for bone mineral density (BMD) to verify OP status. Then bone defects were made in the proximal metaphysis of the tibiae and autologous iliac crest grafts with simultaneous implant placement were performed. The animals were killed at 8 and 12 weeks after bone graft surgery. Undecalcified sections were prepared and examined histologically and histomorphometrically. RESULTS: Osteoporotic status caused by ovariectomy was verified by significantly decreased BMD in experimental group (P < 0.001). At 8 and 12 weeks after bone graft surgery, osseointegration was observed in both groups. However, thickness of cortical bone (TCB), bone volume in cancellous area (BVC), implant-bone contact rate (IBCR) at bone graft area all significantly decreased in experimental group when compared with control group (P < 0.01). Newly formed bone was also less in experimental group than that in control group. CONCLUSION: Although experimental OP may not delay osseointegration of dental implants in autologous iliac crest graft, it certainly promotes resorption of bone grafts, decreases cancellous bone volume and implant-bone contact rate. Therefore it may be an important risk factor for patients receiving autologous bone graft with simultaneous implant placement.

[Response of bone marrow mesenchymal stem cells to mechanical stretch and gene expression of transforming growth factor-beta and insulin-like growth factor-II under mechanical strain].

OBJECTIVE: To study the response of rat bone marrow mesenchymal stem cells (MSCs) to a single period of mechanical strain and expression patterns of transforming growth factor-beta (TGF-beta) and insulin-like growth factor-II (IGF-II) after mechanical stretch. METHODS: Bone marrow MSCs were isolated from SD rats and cultured in vitro. A four-point bending apparatus were used to perform a single period of mechanical strain (2000 microepsilon, 40 min) on MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity of MSCs were examined and gene expression patterns of TGF-beta and IGF-II were detected by SYBR green quantitative real-time RT-PCR. RESULTS: Cell proliferation, ALP activity and expression of TGF-beta and IGF-II were all significantly up-regulated in stretched MSCs when compared with their controls. The mRNA levels of TGF-beta and IGF-II got top increase immediately after mechanical loading and increased about 51.44 and 8.92 folds, respectively, when compared with control cells. Expression of TGF-beta and IGF-II decreased with time and returned to control level at 12 h after mechanical stimulus, despite of a small increase at 6 h. CONCLUSION: The mechanical stretch can promote MSCs proliferation, up-regulate its ALP activity and induce a time-dependent expression increase of TGF-beta and IGF-II which in turn result in osteogenic differentiation of MSCs. Mechanical stimulus is a key stimulator for osteogenic differentiation of MSCs and vital for bone formation in distraction osteogenesis.

Expression of bone-related genes in bone marrow MSCs after cyclic mechanical strain: implications for distraction osteogenesis.

AIM: Understanding the response of mesenchymal stem cells (MSCs) to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis. METHODOLOGY: In this study, a single period of cyclic mechanical stretch (0.5 Hz, 2,000 microepsilon) was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity was examined. The mRNA expression of six bone-related genes (Ets-1, bFGF, IGF-II, TGF-beta, Cbfa1 and ALP) was detected using real-time quantitative RT-PCR. RESULTS: The results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfa1 expression became elevated later. The temporal expression pattern of ALP coincided perfectly with Cbfa1. CONCLUSION: The results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.

Autogenous standard versus inside-out vein graft to repair facial nerve in rabbits.

OBJECTIVE: To evaluate autogenous vein grafts and inside-out vein grafts as conduits for the defects repair in the rabbit facial nerves. METHODS: The 10 mm segments of buccal division of facial nerve were transected for 48 rabbits in this study. Then the gaps were immediately repaired by autogenous vein grafts or inside-out vein grafts in different groups. All the animals underwent the whisker movement test and electrophysiologic test during the following 16 weeks at different time points postoperatively. Subsequently, the histological examination was performed to observe the facial nerve regeneration morphologically. RESULTS: At 8 weeks after operation, the facial nerve regeneration has significant difference between the experimental group and the control group in electrophysiologic test and histological observation. However, at the end of this study, 16 weeks after operation, there was no significant difference between inside-out vein grafts and standard vein grafts in enhancing peripheral nerve regeneration. CONCLUSION: This study suggest that both kinds of vein grafts play positive roles in facial nerve regeneration after being repaired immediately, but the autogenous inside-out vein grafts might accelerate and facilitate axonal regeneration as compared with control.

[Osteogenic differentiation of rat bone marrow mesenchymal stem cell after transfection with recombinant pAd-bone morphogenetic protein-7].

OBJECTIVE: To study the effect of recombinant pAd-BMP-7 on osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSC). METHODS: Recombinant pAd-bone morphogenetic protein (BMP) 7 was constructed and the titer of recombinant adenovirus was determined. pAd-BMP-7 and pAdTrack-CMV were used to transfect rat MSC. Transfection efficiency was measured by fluorescent microscope and BMP-7 expression was detected by RT-PCR and immunocytochemical analysis. The MSC were then randomly divided into 3 groups: group A received pAd-BMP-7 transfection, group B was transfected with pAdTrack-CMV, and group C received pAdTrack-CMV transfection plus bone supplements. Osteogenic differentiation of MSC was evaluated by examination of mineralization nodes formation. RESULTS: The titer of pAd-BMP-7 reached about 2.0 x 10(15) pfu/L and transfection efficiency of exogenous gene was nearly 99% at day 2. The expression of exogenous gene sustained about 5 to 7 weeks, with a higher level during first 3 weeks. After transfection, transcription of BMP-7 and expression of BMP-7 protein were also verified in MSC. Compared with the negative results in group B, mineralization nodes were formed in both group A and group C. However, group A showed better formation of mineralization nodes than group C (P < 0.01). CONCLUSIONS: The results of this study indicated that recombinant pAd-BMP-7 can successfully transfect rat MSC and accelerate their osteogenic differentiation. The technique explored in this study provides a unique and valuable gene engineering approach for reconstruction of craniofacial bone defects.

[The changes of cytoskeleton F-actin in rat bone marrow mesenchymal stem cells and calvarial osteoblasts under mechanical strain].

OBJECTIVE: To explore the response of rat bone marrow mesenchymal stem cells (MSCs and calvarial osteoblasts to mechanical strain and the consequent changes of cytoskeleton F-actin. METHODS: Bone marrow MSCs and calvarial osteoblasts were isolated from SD rats and cultured in vitro. Mechanical stretch was performed on passage 3 cells at 2 000 microepsilon for 0, 2, 6 and 12 hours using four-point bending system. The response of cells and the distribution of F-actin were observed using fluorescent staining under laser scanning confocal microscope and the morphological parameters were quantified using image analysis software Laserpix. RESULTS: Under mechanical stretch, the fluorescent staining decreased obviously at both MSCs and osteoblasts, and F-actin filaments were rearranged and became tenuous, thinner, and abnormally distributed. The outline of nucleus became unclear and apoptotic changes were observed at some of both cells. Cellular size decreased more significantly in MSCs than in osteoblasts. Quantity analysis showed that total area of cells, total fluorescent density and green fluorescent density (F-actin) were all significantly decreased in MSCs (P < 0.05 or P < 0.01), and total fluorescent density, green fluorescent density and red fluorescent density (nuclei) did also in osteoblasts (P < 0.05 or P < 0.01). CONCLUSION: Mechanical stretch caused extensive response on both MSCs and osteoblasts which led to the rearrangement of F-actin filament and apoptosis in some of these cells. MSCs were more sensitive to mechanical strain than osteoblasts.

[Construction of recombinant plasmid pEGFP-BMP7 and its expression in rat bone marrow mesenchymal stem cells in vitro].

OBJECTIVE: To construct recombinant plasmid pEGFP-BMP7 and determine its expression in rat bone marrow mesenchymal stem cells (MSCs) in vitro. METHODS: cDNA of target gene was obtained from neonatal rat kidney by RT-PCR. After sequencing the target gene, the cDNA was subcloned into a eukaryote plasmid pEGFP-N1 by directed cloning and then digested with two restrictive endonucleases to verify the correctiveness of the recombinant plasmid pEGFP-BMP7. Rat bone marrow MSCs were transiently transfected with the pEGFP-BMP7 and transfection efficiency of the Green Fluorescent Protein (GFP) was determined. RT-PCR and immunocytochemical analysis were also performed to detect the expression of BMP7 in rat MSCs. RESULTS: 1 311 bp cDNA fragment was obtained by RT-PCR and sequence analysis showed it matched perfectly with that of rat BMP7 gene except a single nucleotide change at 756 bp from T to A. Digestion of the recombinant plasmid showed two 1.3 kb and 4.7 kb fragments and their size were same as those of BMP7 and pEGFP. This indicated that BMP7 cDNA was successfully subcloned into pEGFP. Transient transfection showed an efficiency of 33% at day 2 in rat MSCs. After transfection, transcription of BMP7 was detected in MSCs and expression of BMP7 protein was also verified. CONCLUSION: Recombinant eukaryote plasmid pEGFP-BMP7 was successfully constructed and expressed in rat bone marrow MSCs. This procedure may provide a unique method for stimulation of callus formation in distraction osteogenesis and reconstruction of craniofacial bone defects.