During Distraction Osteogenesis Research Articles

H Vessel Formation as a Marker for Enhanced Bone Healing in Irradiated Distraction Osteogenesis.

In the setting of bone defects, the injured vasculature and loss of hemodynamic inflow leads to hematoma formation and low oxygen tension which stimulates vascular expansion through the HIf-1α pathway. Most importantly, this pathway upregulates sprouting of type H vessels (CD31hiEmcnhi vessels). H vessels engage in direct interaction with perivascular osteoprogenitor cells (OPCs), osteoblasts, and preosteoclasts of bone formation and remodeling. This angiogenic-osteogenic coupling leads to synchronous propagation of vascular and bony tissue for regenerative healing. A growing body of literature demonstrates that H vessels constitute a large portion of bone's innate capacity for osteogenic healing. We believe that CD31hiEmcnhi vessels play a role in bone healing during distraction osteogenesis (DO). DO is a procedure that utilizes traction forces to facilitate induction of endogenous bone formation and regeneration of surrounding soft tissues such as skin, muscle, tendon, and neurovascular structures. While the H vessel response to mechanical injury is adequate to facilitate healing in normal healthy tissue, it remains inadequate to overcome the devastation of radiation. We posit that the destruction of CD31hiEmcnhi vessels plays a role in precluding DO's effectiveness in irradiated bone defect healing. We aim, therefore, to recapitulate the normal pathway of bony healing by utilizing the regenerative capacity of H vessels. We hypothesize that using localized application of deferoxamine (DFO) will enhance the H vessel-mediated vasculogenic response to radiation damage and ultimately enable osteogenic healing during DO. This discovery could potentially be exploited by developing translational therapeutics to hopefully accelerate bone formation and shorten the DO consolidation period, thereby potentially expanding DO's utilization in irradiated bone healing. Sprague-Dawley rats were divided into three groups: DO, radiation with DO (xDO), and radiation with DO and DFO implantation (xDODFO). Experimental groups received 35 Gy of radiation. All groups underwent DO. The treatment group received injections into the osteotomy site, every other day, beginning on postoperative day (POD) 4 of DFO. Animals were sacrificed on POD 40. For immunohistochemical analysis, mandibles were dissected and fixed in 4% paraformaldehyde for 48 hours, decalcified in Cal-Ex II for 2 days, dehydrated through graded ethanol of increasing concentration, and then embedded in paraffin. Samples were cut into 7-μm thick longitudinally oriented sections including the metaphysis and diaphysis. CD31 and Emcn double immunofluorescent staining were performed to evaluate the extent of CD31hiEmcnhi vessel formation. Bone sections were then stained with conjugated antibodies overnight at 4°C. Nuclei were stained with Hoechst. Slides were also double stained with Osterix and CD31 to study the quantity of H vessel-mediated recruitment of OPCs to accelerate bone healing. Images were acquired with a Nikon Ti2 widefield microscope and analyzed in NIS- Elements Advanced Research 5.41.02 software. The abundance of type H vessels is represented by the area fraction of CD31 + Emcn+ vessel area inside the regenerate sample. OPC concomitant proliferation into the distraction gap is represented by the area fraction of Osterix+ cell area inside of the regenerate sample. There were 6× more type H vessels in DO groups than in xDO groups. Localized DFO significantly increased the abundance of type H vessels of irradiated DO animals compared to xDO by 15× ( p = 0.00133531). Moreover, the DO and xDODFO groups with higher abundance of type H vessels also demonstrated better angiogenesis and osteogenesis outcomes. Interestingly, xDODFO groups doubled the quantity of H vessel formation compared to DO, indicating a supraphysiologic response ( p = 0.044655055). Furthermore, H vessel-mediated recruitment of OPCs mimicked the described H vessel formation trend in our study groups. Irradiated DO groups contained 3× less OPCs compared to DO controls. DFO treatment to xDO animals remediated irradiation damage by containing 12× Osterix+ cells. Finally, DFO treatment of irradiated animals quadrupled osteoprogenitor recruitment into the distraction gap compared to DO controls. In this study, we developed a novel approach to visualize CD31hiEmcnhi in paraffin sections to study DO regeneration. Normal DO demonstrated a significant upregulation of H vessel formation and associated angiogenic-osteogenic coupling. Radiation severely decreased H vessel formation along with an associated significant diminution of new bone formation and nonunion. DFO administration, however, resulted in vascular replenishment and the restoration of high quantities of CD31hiEmcnhi and OPCs, recapitulating the normal process of bony regeneration and repair. DFO treatment remediated new bone formation and bony union in irradiated fields associated with increased H vessel angiogenic-osteogenic coupling. While further studies are required to optimize this approach, the results of this study are incredibly promising for the long-awaited translation of localized DFO into the clinical arena.

LATS1/YAP1 Axis Controls Bone Regeneration on Distraction Osteogenesis by Activating Wnt/β-Catenin.

The Hippo signaling pathway inhibits cell growth, and its components and functions are highly conserved in mammals. LATS1 is a core component of the Hippo signaling pathway associated with lymphatic invasion, astrogliosis, apoptosis, and autophagy. Nevertheless, the role of Hippo/LATS1 in osteogenesis remains unclear. In this study, we used ribonucleic acid (RNA) lentiviruses to inhibit the expression of Lats1 in bone marrow-derived stem cells (BMSCs) and distraction osteogenic regions in rats. Increased osteogenic, proliferative, and migratory abilities of BMSCs were observed in Lats1-inhibited BMSCs, while these phenotypes were partially reversed by YAP1 inhibition. In vivo, we found that the LATS1/YAP1 axis promoted osteogenesis during distraction osteogenesis (DO). β-catenin was positively correlated with YAP1 expression in vivo and in vitro. When YAP1 was strongly positive in the nucleus, β-catenin expression was upregulated; when YAP1 expression was inhibited by verteporfin, β-catenin was not expressed in the nucleus. These findings suggest that the LATS1/YAP1 signaling axis promotes DO by activating the Wnt/β-catenin signaling pathway. This study provides insights into the molecular mechanism of osteogenesis and a potential therapeutic strategy for bone regeneration in DO by associating with LATS1/YAP1-β-catenin.

A comparative study of bone union and nonunion during distraction osteogenesis

BackgroundThe clinical characteristics of bone nonunion during distraction osteogenesis (DO) were rarely discussed. This study was employed to specify the difference between bone union and nonunion during DO.MethodsThe patients with bone lengthening were recruited in our study. The bone union cases indicated the ones that remove the external fixator successfully, whereas the bone nonunion represented the bridging callus did not appear even after 9 months (an absence of bridging callus for at least three out of four cortices on plain radiographs) that needs autogenous bone transplantation. The differences in the pixel value ratio (PVR) growth of regenerated callus, lengthening index (LI), healing index (HI), external fixation index (EFI) and blood biochemical indexes between bone union and nonunion were analyzed.ResultsA total of 8 bone nonunion and 27 bone union subjects were included in this study. The PVR growth in bone nonunion was significantly lower than that in bone union (0.19 ± 0.06 vs. 0.32 ± 0.16, P = 0.048). Interestingly, the HI and EFI in bone nonunion was significantly higher than that in bone union (62.0 ± 31.4 vs. 37.0 ± 27.4, P = 0.036; 75.0 ± 30.9 vs. 49.9 ± 16.1, P = 0.006). However, no significant difference with regard to LI was identified (0.76 ± 0.52 vs. 0.77 ± 0.32, P = 0.976). Moreover, the circulating level of urea and lymphocyte count in bone union was significantly lower than that in bone nonunion (4.31 ± 1.05 vs. 5.17 ± 1.06, P = 0.049; 2.08 ± 0.67 vs. 2.73 ± 0.54, P = 0.018). On the contrary, the circulating level of magnesium in bone union was significantly higher than that in bone nonunion (0.87 ± 0.07 vs. 0.80 ± 0.07, P = 0.014).ConclusionCompared to the bone union, the PVR growth was significantly lower, whereas the HI and EFI was significantly higher in the bone nonunion. Moreover, the circulating level of urea, magnesium and lymphocyte count was also different between these two. Therefore, the PVR, HI and EFI seems to be reliable and sensitive indicators to reflect the bone nonunion during DO, which might be considered in bone lengthening. Further prospective studies are still needed to elaborate the concerned issues.

Total flavonoids of Rhizoma Drynariae enhances CD31hiEmcnhi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis byactivating PDGF‑BB/VEGF/RUNX2/OSX signaling axis.

Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney-tonifying Traditional Chinese medicine Rhizoma Drynariae, can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31hiEmcnhi during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31hiEmcnhi vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co-immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31hiEmcnhi vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet-derived growth factor (PDGF)-BB, VEGF, runt-related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription-quantitative PCR. The in vivo assays demonstrated that TFRD markedly promoted CD31hiEmcnhi vessel formation during DO, whereas PDGF-BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF-BB. The protein and mRNA levels of PDGF-BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF-BB. Thus, TFRD could facilitate CD31hiEmcnhi vessel formation and subsequently enhance angiogenic-osteogenic coupling to regenerate bone defects during DO via the PDGF-BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31hiEmcnhi vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31hiEmcnhi vessels.

Pain levels during distraction osteogenesis with lengthening nails in 168 cases

PurposeTo firstly examine the pain levels during distraction osteogenesis (DO) with lengthening nails (LN) in a large sample.MethodsA total of 168 cases underwent DO of the tibia or femur with five different models of LN. Under a standardized medical regime, daily pain levels were noted as nominal rating scale (NRS) score (0–10) during the distraction phase. NRS scores and several potential influence factors (LN model, bone, approach, side, age, gender) were evaluated.ResultsThe mean distraction length was 39.1 ± 14.4 mm. The average NRS score decreased from postoperative day 1 with 2.84 nonlinearly by 1.03 points (36.3%) over the course of 62 days to an average score of 1.81. The mean decrease during the first thirty days was 0.67(23.6%). Subgroup analysis did not reveal any influence factors.ConclusionPain levels during the distraction phase are overall low, continuously decreasing, and well manageable with mostly non-opioid analgesics.

Effect of bone morphogenic protein-2 and desferoxamine on distraction osteogenesis

BackgroundAngiogenesis is crucial for formation of a stable regenerate during distraction osteogenesis (DO). This experimental study evaluates if bone morphogenic protein-2 (BMP-2) and desferrioxamine (DFO), two agents which are known to induce neoangiogenesis in vivo, would increase angiogenesis and osteogenesis, and improve mechanical properties of bone regenerate in DO model. MethodsTwenty-four tibias of 24 New Zealand rabbits were osteotomized and fixed with semi-circular fixators. Three groups of 8 animals were formed. BMP-2 soaked scaffolds were used in the first group, whereas daily local DFO injections were made in the second group. Subjects in the control group did not receive any agents during the surgery or in the distraction period. The rabbits in all three groups underwent distraction at a rate of 0.6 mm/day for 15 days following the 7-day latent period. Animals were sacrificed on day 38, and the tibia were harvested for histological and mechanical examination of the regenerate. ResultsAll 24 rabbits survived the surgical procedure, and there were no side effects against the BMP-2 and local DFO. Three-point bending tests revealed a higher force (361 ± 267 N.) required for fracture in Group 1 (p: 0.018). Similarly, the bending moment in Group 1 (5.4 ± 4.0 Nmm) was significantly higher than the other groups (p: 0.021). There was no significant difference between the groups in terms of deflection and stiffness (p ˃ 0.05). Histologically, there was no statistical difference between the groups in terms of endochondral, periosteal, and intramembranous ossification and VEGF activity (p ˃ 0.05). ConclusionBMP-2 and DFO stimulate angiogenesis by increasing VEGF activity. Angiogenesis is one of the most important mechanisms for the initiation and maintenance of new bone formation. Stimulation of angiogenesis in unfavorable biomechanical conditions may not be sufficient for ideal bone formation.

Hypoxia During the Consolidation Phase of Distraction Osteogenesis Promotes Bone Regeneration.

BackgroundHypoxia is the critical driving force for angiogenesis and can trigger the osteogenic-angiogenic coupling followed by the enhancement of bone regeneration. While lots of studies showed that hypoxia administration can accelerate bone formation during distraction osteogenesis (DO), the therapeutic timing for the osteogenic purpose was concentrated on the distraction phase. The outcomes of hypoxia administration in the consolidation phase stay uncertain. The purpose of this study was to determine the osteogenic effectiveness of hypoxia therapy during the consolidation phase, if any, to enhance bone regeneration in a rat femoral DO model.MethodsA total of 42 adult male Sprague-Dawley rats underwent right femoral mid-diaphysis transverse osteotomy and were randomly divided into Control (NS administration, n = 21) and Group1 (deferoxamine therapy, n = 21) after distraction. During the consolidation phase, Group1 was treated with local deferoxamine (DFO) injection into the distraction zone, while the Control underwent the same dosage of NS. Animals were sacrificed after 2, 4, and 6 weeks of consolidation. The process of bone formation and remodeling was monitored by digital radiographs, and the regenerated bone was evaluated by micro-computed tomography (micro-CT), biomechanical test, and histological analysis. The serum content of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) were measured by enzyme linked immunosorbent assay (ELISA) for further analysis.ResultsBone regeneration was significantly enhanced after hypoxia therapy during the consolidation phase. The digital radiograph, micro-CT, and biomechanical evaluation showed better effects regarding volume, continuity, and mechanical properties of the regenerated bone in Group1. The histomorphological evaluation also revealed the hypoxia treatment contributed to accelerate bone formation and remodeling during DO. The higher positive expression of angiogenic and osteogenic markers were observed in Group1 after hypoxia administration according to the immunohistochemical analysis. The serum content of HIF-1α and VEGF was also increased after hypoxia therapy as evidenced from ELISA.ConclusionHypoxia administration during the consolidation phase of distraction osteogenesis has benefits in enhancing bone regeneration, including accelerates the bone formation and remodeling.

Cyclic Distraction-Compression Dynamization Technique Enhances the Bone Formation During Distraction Osteogenesis.

Background: Interfragmentary movements have benefits in the improvement of bone formation during distraction osteogenesis (DO). Although several clinical studies reported positive outcomes regarding the application of the cyclic distraction–compression (CDC) dynamization technique in cases with poor bone formation during DO, they are mostly anecdotal without a detailed description. The purpose of this study was to investigate the effectiveness and potential mechanism of different amplitudes and rates of the CDC technique on bone regeneration in a rat femur DO model. Methods: A total of 60 adult male Sprague-Dawley rats underwent right femoral mid-diaphysis transverse osteotomy and were randomly and evenly divided into Control (no manipulation), Group1 (CDC therapy), Group2 (CDC therapy with larger amplitude), and Group3 (CDC therapy with a slower rate) after distraction. The CDC technique was performed during the middle phase of the consolidation period according to different protocols. Animals were sacrificed after 4 and 6 weeks of consolidation. The process of bone formation was monitored by digital radiographs, and the regenerate bone was evaluated by micro-computed tomography (micro-CT), biomechanical test, and histological analysis. The serum contents of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were measured by enzyme-linked immunosorbent assay (ELISA). Results: Bone regeneration after the CDC technique was improved significantly during DO. The digital radiograph, micro-CT, histomorphological analysis, and biomechanical evaluation showed better effects regarding volume, continuity, and mechanical properties of the regenerate bone in Group2 and Group3 when compared to Group1. The angiogenic and osteogenic markers were more highly expressed in Group2 and Group3 than in Group1 according to the immunohistochemical analysis. As for ELISA, the serum contents of HIF-1α and VEGF were also increased after the CDC technique, especially in Group2 and Group3. Conclusion: The CDC dynamization technique has benefits on the improvement of bone formation during DO, and the mechanism may be due to tissue hypoxia activating the HIF pathway followed by the augmentation of osteogenic–angiogenic coupling. Better outcomes may be achieved by moderately increasing the amplitude and slowing down the rate of the CDC technique.

Accelerated Bone Regeneration by Adrenomedullin 2 Through Improving the Coupling of Osteogenesis and Angiogenesis via β-Catenin Signaling.

Both osteogenic differentiation and the pro-angiogenic potential of bone marrow mesenchymal stem cells (BMSCs) contribute to bone regeneration during distraction osteogenesis (DO). Adrenomedullin 2 (ADM2), an endogenous bioactive peptide belonging to the calcitonin gene-related peptide family, exhibits various biological activities associated with the inhibition of inflammation and the attenuation of ischemic-hypoxic injury. However, the effects and underlying mechanisms of ADM2 in osteogenic differentiation and the pro-angiogenic potential of BMSCs, along with bone regeneration, remain poorly understood. In the present study, we found that osteogenic induction enhanced the pro-angiogenic potential of BMSCs, and ADM2 treatment further improved the osteogenic differentiation and pro-angiogenic potential of BMSCs. Moreover, the accumulation and activation of β-catenin, which is mediated by the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the activation of protein kinase B (AKT), have been shown to contribute to the effects of ADM2 on BMSCs. In vivo, ADM2 accelerated vessel expansion and bone regeneration, as revealed by improved radiological and histological manifestations and the biomechanical parameters in a rat DO model. Based on the present results, we concluded that ADM2 accelerates bone regeneration during DO by enhancing the osteogenic differentiation and pro-angiogenic potential of BMSCs, partly through the NF-κB/β-catenin and AKT/β-catenin pathways. Moreover, these findings imply that BMSC-mediated coupling of osteogenesis and angiogenesis may be a promising therapeutic strategy for DO patients.

Accelerated Bone Regeneration by Astragaloside IV through Stimulating the Coupling of Osteogenesis and Angiogenesis.

Both osteoblasts and preosteoclasts contribute to the coupling of osteogenesis and angiogenesis, regulating bone regeneration. Astragaloside IV (AS-IV), a glycoside of cycloartane-type triterpene derived from the Chinese herb Astragalus membranaceus, exhibits various biological activities, including stimulating angiogenesis and attenuating ischemic-hypoxic injury. However, the effects and underlying mechanisms of AS-IV in osteogenesis, osteoclastogenesis, and bone regeneration remain poorly understood. In the present study, we found that AS-IV treatment inhibited osteoclastogenesis, preserved preosteoclasts, and enhanced platelet-derived growth factor-BB (PDGF-BB)-induced angiogenesis. Additionally, AS-IV promoted cell viability, osteogenic differentiation, and angiogenic gene expression in bone marrow mesenchymal stem cells (BMSCs). The activation of AKT/GSK-3β/β-catenin signaling was found to contribute to the effects of AS-IV on osteoclastogenesis and osteogenesis. Furthermore, AS-IV accelerated bone regeneration during distraction osteogenesis (DO), as evidenced from the improved radiological and histological manifestations and biomechanical parameters, accompanied by enhanced angiogenesis within the distraction zone. In summary, AS-IV accelerates bone regeneration during DO, by enhancing osteogenesis and preosteoclast-induced angiogenesis simultaneously, partially through AKT/GSK-3β/β-catenin signaling. These findings reveal that AS-IV may serve as a potential bioactive molecule for promoting the coupling of osteogenesis and angiogenesis, and imply that AKT/GSK-3β/β-catenin signaling may be a promising therapeutic target for patients during DO treatment.

Wire bending development and progression during Ilizarov system tibial lengthening in skeletally immature patients with achondroplasia

Summary Background Tensile strength develops on wires during distraction osteogenesis (DO) using the Ilizarov system. The increase of tensile strength can lead to bending of the wires. The purpose of this study was to investigate the development and progression of wire bending during tibial lengthening using the Ilizarov system. We also identified factors that contribute to wire bending. Methods Seventy-two segments of 36 skeletally immature achondroplasia patients (mean age, 8.5 years; range, 5.1–14.4 years) with bilateral tibial lengthening with the Ilizarov system were included in this study. The wire bending was defined as cases with loss of linearity of the wire during DO. The average distance of proximal and distal wires to the adjacent ring throughout the entire DO was measured to determine the progression of wire bending. In addition, age, height, weight, initial tibial length, gain of length, amount of correction of angular deformity, distraction index, external fixation index, and healing index were investigated to identify potential contributors to wire bending. Wire-related complications, including wire breakage, were also assessed. Results Wire bending increased in the distraction phase (p ​ Conclusion This study indicated that the wire bending, which represented tensile strength on the wire, changed during the course of the DO. In addition, the amount of lengthening and the patient’s weight may contribute to the degree of the wire bending. The translational potential of this article Because excessive wire bending could lead to wire-related pain or even wire breakage, it is necessary to finely check and reduce the tension of the wire during DO in particular case of heavy patients or patients undergoing large amount of lengthening. Nevertheless, in case of wire breakage, remove the broken wire or fix it using the additional small plate to the Ilizarov system.

Bone regeneration and mineralisation was promoted during distraction osteogenesis by human periostin gene in rabbit mandibular model

ObjectiveTo study the effect of periostin gene on bone regeneration and mineralisation during distraction osteogenesis (DO) in a rabbit mandibular model. Subjects and methodsA total of 54 New Zealand white rabbits underwent mandibular lengthening and were divided into group A and group B (n=27, each). At the end of the distraction, adenoviruses containing human POSTN/enhanced green fluorescent protein (Ad5-POSTN/EGFP) viruses or Ad5-POSTN viruses were injected into the distraction gaps in the group B or A, respectively. On the 4th and 8th week after distraction, eight samples, randomly selected from each group, were subjected to CT scanning and dual energy X-ray absorptiometry detection. All animals from each group were sacrificed for micro-CT, histology, and three-point bend testing eight weeks after DO. ResultsThe CT images showed that callus in the distraction gap of group B was significantly better than that of group A. On the 4th and 8th weeks, the bone mineral densities (BMD) of the bone tissue in the distraction gap of the group B were 0.163±0.011g/cm2 and 0.239±0.024g/cm2, respectively, while the bone mineral contents (BMC) was 0.099±0.011g and 0.205±0.018g, respectively, which were significantly higher compared with group A. Histological sections showed that the trabecular bone in the distraction gap of group B was more coarse. On the basis of micro-CT images and micro-architecture parameters show that the microstructure of the distraction gap of the group B had more mature characteristics. The biomechanical strength of the distracted gap in the group B was 234.30±30.48N, which was 1.28 times higher than that in the group A (P=0.003). ConclusionLocal transduction with human periostin gene would promote osteogenesis and mineralisation in a rabbit mandibular DO model. Periostin might be considered for clinical use if only there is a safe and effective administration method.

Low-intensity pulsed ultrasound enhances callus consolidation in distraction osteogenesis of the tibia by the technique of lengthening over the nail procedure

BackgroundLow-intensity pulsed ultrasound (LIPUS) has been widely accepted in promoting the fracture healing process. However, there have been limited clinical trials focused on the efficacy of LIPUS during distraction osteogenesis (DO) by the technique of lengthening over the nail procedure. The purpose of the current study was to evaluate the efficacy of LIPUS during DO.MethodsWe retrospectively evaluated 30 patients (60 segments) who underwent simultaneous bilateral tibial lengthening over the nail. The patients were grouped into the LIPUS group and the control group based on LIPUS stimulation. The two patient groups were compared for demographic data (sex, age at operation, preoperative height, BMI, and smoking history), qualitative assessments of the callus (callus shape and type), external fixation index, and four cortical healing indexes.ResultsFifteen patients (30 segments) were classified as the LIPUS group, and another 15 patients (30 segments) were classified as the control group. No significant differences were found in the assessed demographic data between the groups. LIPUS stimulated a more cylindrical, more homogenous, and denser type of callus formation at the end of the distraction phase. The two groups exhibited equivalent outcomes in terms of external fixation index (p = 0.579). However, significant differences were found in healing indexes of the anterior and medial cortices (p < 0.001 and p = 0.002, respectively). The healing indexes of those cortices in the LIPUS group (mean of 36.6 days/cm and 32.5 days/cm, respectively) reflected their significantly faster healing compared to the control group (mean HI of 57.5 days/cm and 44.2 days/cm, respectively). There were no LIPUS-related complications.ConclusionsLIPUS is a noninvasive and effective adjuvant therapy to enhance callus maturation during DO. It enhances callus consolidation and may have a positive effect on the appropriate callus shape and type.

Effect of Laser Bio-Stimulation on Mandibular Distraction Osteogenesis: An Experimental Study

The aim of this study was to evaluate the osseous response to laser bio-stimulation clinically and histologically during distraction osteogenesis (DO) induced in the mandibles of mongrel dogs. Thirty dogs were divided into 3 groups of 10 (5 with and 5 without laser treatment) according to sacrifice periods (2, 4, and 8weeks after distraction). DO was performed between the mandibular second and third premolars using an internal linear distractor. After a 7-day latency period, the distractor was activated at the rate of 1mm per day for 10days followed by a consolidation period during which the right mandibular side was irradiated with a diode laser (wavelength [λ], 970nm; power, 2W; spot size, 320μm; total energy [E], 840J), whereas the control group was not irradiated, after distraction. Histologic specimens were prepared and histomorphometric analysis of specimens was performed. Clinical examinations showed that the low-intensity laser diode had a pronounced effect on the quality and quantity of newly formed bone in the DO regenerate in the laser groups compared with the control groups. Histopathologic sections from laser groups I, II, and III displayed the bio-stimulatory effect of laser on new bone through an increased rate of osteoblast proliferation and differentiation, an accelerated rate of intramembranous ossification, and increased neoangiogenesis compared with the control groups. Moreover, the histomorphometric results showed that mean bone trabecular size, bone trabecular total area, and bony area fraction of the regenerate were larger and statistically significant (P<.05) especially in laser groups I and II (early stages of bone formation) compared with the control groups. The low-level diode laser had a positive role as a potential bio-stimulator and local inducer in enhancing bone formation during DO and resulted in early stability of the bone regenerate, a shorter total treatment time, and improved new bone quality and quantity.

The expression of endogenous hydrogen sulfide signal during distraction osteogenesis in a rabbit model

The hydrogen sulfide (H2S) signal system plays an important role in bone metabolism. However, the role of endogenous H2S during distraction osteogenesis (DO) remains unclear. Sixty-two male New Zealand White rabbits were subjected to right mandibular DO. Before distraction, the animals were divided randomly into two groups: group A, 0.5mm twice/day for 10 days; group B, 1.25mm twice/day for 4 days. Plasma and distraction gap tissue were harvested to determine the H2S signal. The osteogenesis effect was also evaluated. The newly regenerated bone in group A presented a higher level of mineralization and biomechanical strength than that in group B. The bone mineralization density in group A was 1.95-fold that in group B (P=0.028), while the biomechanical strength in group A was 1.26-fold that in group B (P=0.042) at the end of the experiment. The H2S signal was detected during the whole process of DO. The relative plasma H2S concentrations in group A were noticeably higher than those in group B at the middle of distraction (P<0.001), at the end of the distraction (P=0.034), and 2 weeks after the end of distraction (P=0.002). The results suggest that the endogenous H2S signal system plays a major role during DO.

Effect of gene transfection timing on TGF-β1 expression in rabbit mandibular distraction gap.

Transforming growth factor-β1 (TGF-β1) is a member of the TGF-β superfamily, and plays an important role in promoting various stages of intramembranous and endochondral bone formation. It is one of the major growth factors that influence new bone formation in the distraction gap during distraction osteogenesis (DO). The major problem of DO is the time required for the treatment. Reports show that gene therapy accelerates osteogenesis, which can significantly benefit patients with DO. However, the optimal timing of gene transfection has not yet been reported. In this study, we used the New Zealand rabbit mandibular DO model for transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 during the latency, distraction, and consolidation periods of DO. The TGF-β1 levels in the distraction gap were detected at different time-points by immunohistochemistry and analyzed semi-quantitatively with the CMIAS-2001A computerized image analyzer. The TGF-β1 levels peaked after 7 days and decreased after 14 days of consolidation in each group. In contrast, the TGF-β1 levels in the transfected distraction period group were significantly higher than those in the other groups. After 28 days of consolidation, TGF-β1 levels decreased and there was no significant difference among the groups. These results indicated that the genes transfected in the distraction period up-regulated the expression of TGF-β1 more than in the latency and consolidation periods, which promoted bone formation in the distraction gap through a series of biological effects. Thus, we obtained a remarkable effect on new bone formation, and showed that the distraction period is optimal for gene therapy.

Scaffold-Based Delivery of Bone Marrow Mesenchymal Stem Cell Sheet Fragments Enhances New Bone Formation In Vivo

Stem cell therapy is becoming a potent strategy to shorten the consolidation time and reduce potential complications during distraction osteogenesis (DO). However, the conventional local injection or scaffold-based delivery of bone marrow mesenchymal stem cell (BMSC) suspension deprives the cells of their endogenous extracellular matrix, which might dampen cell differentiation and tissue regeneration after implantation. Therefore, in our study, a BMSC sheet was established and was then minced into fragments and loaded onto a hydroxyapatite (HA) scaffold for grafting. The purified and characterized BMSCs were grown into a cell sheet, and bone formation and mineralization capacity, as well as the cell sheet composition, were analyzed. Afterward, the invivo osteogenic ability of cell sheet fragments (CSFs) was evaluated in immunocompromised mouse and rabbit models of DO. The BMSC sheet exhibited higher alkaline phosphatase activity than osteogenic cell suspension cultures. Alkaline phosphatase activity and mineral particles in the cell sheet increased further after osteogenic induction. Moreover, calcium and phosphorus were present only in the osteogenic cell sheet, along with the common elements carbon, oxygen, chlorine, sodium, and sulfur, as indicated by x-ray photoelectron spectroscopy analysis. In a mouse model, the CSF-HA complex was injected subcutaneously. Micro-computed tomography analysis showed that the osteogenic CSF-HA complex led to a considerably higher bone volume than the BMSC-HA or CSF-HA complex. The osteogenic CSF-HA specimens showed increased angiogenesis and deposition of type I collagen compared with the non-osteogenic CSF-HA or BMSC-HA specimens. Moreover, the osteogenic CSF-HA markedly improved bone consolidation and increased bone mass in DO rabbits. Collectively, the incorporation of osteogenic BMSC sheets into HA particles greatly promoted bone regeneration, which offers therapeutic alternatives for DO.

Recombinant human bone morphogenetic protein-2 suspended in fibrin glue enhances bone formation during distraction osteogenesis in rabbits.

IntroductionBone morphogenetic protein-2 (BMP-2) has high potential for bone formation, but its in vivo effects are unpredictable due to the short life time. This study was designed to evaluate the effects of recombinant human (rh) BMP-2 suspended in fibrin on bone formation during distraction osteogenesis (DO) in rabbits.Material and methodsThe in vitro release kinetics of rhBMP-2 suspended in fibrin was tested using an enzyme-linked immunosorbent assay. Unilateral tibial lengthening for 10 mm was achieved in 48 rabbits. At the completion of osteodistraction, vehicle, fibrin, rhBMP-2 or rhBMP-2 suspended in fibrin (rhBMP-2 + fibrin) was injected into the center of the lengthened gap, with 12 animals in each group. Eight weeks later, the distracted callus was examined by histology, micro-CT and biomechanical testing. Radiographs of the distracted tibiae were taken at both 4 and 8 weeks after drug treatment.ResultsIt was found that fibrin prolonged the life span of rhBMP-2 in vitro with sustained release during 17 days. The rhBMP-2 + fibrin treated animals showed the best results in bone mineral density, bone volume fraction, cortical bone thickness by micro-CT evaluation and mechanical properties by the three-point bending test when compared to the other groups (p < 0.05). In histological images, rhBMP-2 + fibrin treatment showed increased callus formation and better gap bridging compared to the other groups.ConclusionsThe results of this study suggest that fibrin holds promise to be a good carrier of rhBMP-2, and rhBMP-2 suspended in fibrin showed a stronger promoting effect on bone formation during DO in rabbits.

The effects of hypoxia-inducible factor (HIF)-1α protein on bone regeneration during distraction osteogenesis: an animal study

The aim of this study was to observe the effect of hypoxia-inducible factor (HIF)-1α on bone regeneration during distraction osteogenesis (DO). Fifty-one New Zealand white rabbits underwent mandibular lengthening with a distraction rate of 2mm/day, and were divided randomly into three groups (17 in each). Group C rabbits received 20μg rHIF-1α, group B received 10μg rHIF-1α, and group A received 100μl saline injection in the distraction gap every day for 10 days. Radionuclide bone imaging (RBI), computed tomography, dual energy X-ray absorptiometry, radiography, histology, and three-point bend testing were performed. RBI showed that the uptake ratio in group B (1.41±0.25, P=0.013) and group C (1.64±0.37, P<0.001) was higher than that in group A (1.01±0.26). The bone mineralization density and bone mineralization content in group C were highest among the three groups. Radiology and histology findings indicated more callus regeneration in groups C and B. Mechanical testing demonstrated that the ultimate force in group C (289.71±43.31N, n=6) was 1.49-fold (P<0.001) that of group A and 1.20-fold (P=0.012) that of group B. HIF-1α may represent a new agent to promote DO by accelerating osteogenesis and mineralization.

Analysis by means of cone-beam computed tomography, bone density and X-ray beam attenuation of rabbit mandibles subjected to low-level laser therapy during distraction osteogenesis

&lt;p&gt;&lt;strong&gt;&lt;em&gt;Objectives: &lt;/em&gt;&lt;/strong&gt;&lt;em&gt;To assess by means of cone-beam computed tomography (CBCT) the relative bone density of newly formed bone in rabbit mandibles treated with low-level laser therapy (LLLT) during distraction osteogenesis (DO). &lt;/em&gt;&lt;strong&gt;&lt;em&gt;Methods: &lt;/em&gt;&lt;/strong&gt;&lt;em&gt;Seven rabbits underwent surgical osteotomy and immediate installation of a distractor on the mandible (right side). DO was performed for 10 days, and LLLT (aluminum-gallium-arsenide [AlGaAs] infrared laser, λ 830 nm, 40 mW) was applied during distractor activation (days 4-10). Three rabbits were euthanized at the end of the activation period (day 10, group A), and four at the end of the maturation period (day 20, group B). Quality and quantity of newly formed bone in the distracted area were measured on grayscale images using adjacente untreated areas as controls. Two CBCT images were acquired for each animal (before and after removal of soft tissue) to evaluate the influence of the soft tissue on X-ray beam attenuation. &lt;/em&gt;&lt;strong&gt;&lt;em&gt;Results: &lt;/em&gt;&lt;/strong&gt;&lt;em&gt;Mean grayscale values in the distracted area were higher in group B rabbits (140.47 vs. 102.55 in group A), indicating greater bone maturation in a short period. Absence of soft tissues during CBCT scanning was associated with higher grayscale values, indicative of less X-ray beam attenuation. &lt;/em&gt;&lt;strong&gt;&lt;em&gt;Conclusions: &lt;/em&gt;&lt;/strong&gt;&lt;em&gt;It is possible to measure differences in bone density in areas subjected to DO on CBCT scans, providing an objective assessment useful for monitoring bone quality during the repair process.&lt;/em&gt;&lt;/p&gt;&lt;p&gt;&lt;em&gt; &lt;/em&gt;&lt;/p&gt;