Osteogenesis and mineralization in a rabbit mandibular distraction osteogenesis model is promoted by the human LMP‐1 gene

To observe the effects of LIM mineralization protein‐1 (LMP‐1) on bone regeneration in the distraction zone based on gene transduction, 36 New Zealand white rabbits underwent mandibular lengthening with a distraction rate of 2 mm/day. The animals were then randomly divided into group A and group B (n = 18, each). At the end of the distraction, Ad5‐EGFP viruses and Ad5‐LMP‐1/EGFP viruses were injected into the distraction gaps in groups A and B, respectively. Seven days later, five randomly selected animals from each group were sacrificed to evaluate the survival of the virus. Four and 8 weeks after distraction osteogenesis (DO), six samples randomly selected from each group underwent CT scanning and dual energy X‐ray absorptiometry detection. Eight weeks after DO, the rabbits were sacrificed, and the distracted mandibles were harvested. Six animals from each group processed for radiography, micro‐CT, histology, and the rest samples were taken three‐point bend testing. Using this model, better bone formation and mineralization in the distracted callus were observed in group B when compared with those in group A. The results suggest local transduction with LMP‐1 gene promotes osteogenesis and mineralization in DO. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:521–526, 2015.     

Callus formation enhanced by BMP-7 ex vivo gene therapy during distraction osteogenesis in rats.

This study was designed to observe the effects of bone morphogenetic protein-7 (BMP-7) ex vivo gene therapy on callus formation during rat mandibular distraction osteogenesis (DO). Fifty-four Sprague-Dawley rats underwent osteodistraction of the right mandible and were then randomly divided into three groups. Immediately after distraction, autologous bone marrow mesenchymal stem cells (MSCs) transfected with BMP-7, MSCs untransfected with BMP-7, and physiological saline were injected into the distraction gaps of the mandibles in groups A, B, and C, respectively. Nine animals from each group were euthanized at 2 and 6 weeks after completion of distraction. The distracted mandibles were removed and processed for radiographic, histological, immunohistochemical, and scanning electron microscopic examinations as well as Ca/P ratio analysis. Group A animals showed greater bone formation and earlier mineralization in the distracted callus when compared with group B. Similarly increased callus formation was found in group B than group C. Positive immunostaining of BMP-7 was observed in the distracted callus in all groups. However, BMP-7 expression was much stronger in group A compared with groups B and C. The results of this study suggest that BMP-7-mediated ex vivo gene transfer based on MSCs may accelerate callus formation in distraction osteogenesis and facilitate consolidation. Local gene therapy may ultimately be an alternative or supplemental approach to DO enhancement, especially for patients whose osteogenic potentials are compromised by diseases such as osteoporosis, severe trauma, and postoncologic irradiation.     

Locally injection of cell sheet fragments enhances new bone formation in mandibular distraction osteogenesis: A rabbit model

Effective methods to shorten the treatment period of distraction osteogenesis (DO) are needed. To investigate whether injections of osteogenic bone marrow stromal cell (BMSC) sheet fragments could be used to facilitate new bone formation during DO, 30 rabbits underwent bilateral mandibular osteotomy and their mandibles were lengthened at a rate of 0.75 mm/12 h for 6 days after a 5‐day latency period. There were three treatment groups (n = 10 for each group): Serum‐free medium, dissociated BMSCs, and BMSC sheet fragments. A local injection was conducted with a needle directly into the distracted areas immediately after distraction. Rabbits were sacrificed for examination at 3 and 6 weeks after injection. Gross examination, radiographic evaluation, and micro‐CT scanning indicated a significant increase in bony union in the BMSC sheet fragment group, compared with the medium group and the dissociated cell group. The histomorphometric analysis showed more intensive bone formation in the sheet fragment group than the other two groups at each time point. Additionally, the peak load was significantly higher in the fragment group than those in the others. The results show that injection of BMSC sheet fragments promotes bone formation in DO and indicate a promising approach to shorten the treatment period of osteodistraction. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1082–1088, 2013     

Serial bone densitometry during mandibular distraction osteogenesis.

The purpose of this study was to obtain serial measurements of bone mineral density (BMD) of the mandible during distraction osteogenesis. Fourteen skeletally mature male rabbits were subjected to unilateral mandibular osteodistraction at a defined rate. Two animals were sacrificed each week after surgery for 7 wk. The mandible and overlying soft tissue were resected and the BMD measured using dual-energy X-ray absorptiometry (DXA). Measurements obtained from the generate bone were compared to the contralateral (control) hemi-mandible. In the control hemi-mandible, BMD remained stable throughout the distraction protocol at 0.5 gm/cm2. In the distracted hemi-mandible, BMD sharply decreased to 0.35 gm/cm2 by the second week of distraction but steadily increased starting the third week of distraction. BMD surpassed control levels by wk 7. BMD measurements may provide a noninvasive assessment of bone mineralization and strength during distraction osteogenesis.     

New bone formation enhanced by ADSCs overexpressing hRunx2 during mandibular distraction osteogenesis in osteoporotic rabbits

Promoting new bone formation during distraction osteogenesis (DO) in elderly patients with osteoporosis is still a challenge. In this study, we investigated the effect of gene therapy using local Runt‐related gene 2 on new bone formation during osteoporotic mandibular DO in rabbits. First, we successfully established a mandibular osteoporotic animal model by ovariectomizing rabbits. Second, the right mandibles of the osteoporotic rabbits were distracted after corticotomy. The distraction gap of the rabbits in Group A2 and B2 were injected with Adv‐hRunx2‐GFP‐transfected adipose‐derived stromal cells (ADSCs) and Adv‐GFP‐transfected ADSCs, respectively. Rabbits in Groups C2 (ovariectomized control) and D2 (sham surgery control) were injected with physiologic saline. New‐generation bone tissue in the distraction gap was analyzed via plain radiographic examinations, micro‐computed tomography, histological examinations, and biomechanical testing at weeks 3, 6, and 9 of the consolidation period. Results of above examinations showed that no ideal new bone formation was observed in Groups B2 and C2, but obvious ideal new bone formation was observed in Group A2 and D2. The results suggested that gene therapy using rhRunx2‐modified ADSCs promoted new bone formation during osteoporotic mandibular DO and effectively compensated for the detrimental effects of systemic osteoporosis on new bone formation. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:709–720, 2014.     

Parathyroid hormone PTH(1–34) increases the volume,mineral content,and mechanical properties of regenerated mineralizing tissue after distraction osteogenesis in rabbits

Background and purpose Parathyroid hormone (PTH) has attracted considerable interest as a bone anabolic agent. Recently, it has been suggested that PTH can also enhance bone repair after fracture and distraction osteogenesis. We analyzed bone density and strength of the newly regenerated mineralized tissue after intermittent treatment with PTH in rabbits, which undergo Haversian bone remodeling similar to that in humans.

Methods 72 New Zealand White rabbits underwent tibial mid-diaphyseal osteotomy and the callus was distracted 1 mm/day for 10 days. The rabbits were divided into 3 groups, which received injections of PTH 25 µg/kg/day for 30 days, saline for 10 days and PTH 25 µg/kg/day for 20 days, or saline for 30 days. At the end of the study, the rabbits were killed and the bone density was evaluated with DEXA. The mechanical bone strength was determined by use of a 3-point bending test.

Results In the 2 PTH-treated groups the regenerate callus ultimate load was 33% and 30% higher, absorbed energy was 100% and 65% higher, BMC was 61% and 60% higher, and callus tissue volume was 179% and 197% higher than for the control group.

Interpretation We found that treatment with PTH during distraction osteogenesis resulted in substantially higher mineralized tissue volume, mineral content, and bending strength. This suggests that treatment with PTH may benefit new bone formation during distraction osteogenesis and could form a basis for clinical application of this therapy in humans.     

An injectable composite material containing bone morphogenetic protein‐2 shortens the period of distraction osteogenesis in vivo

To investigate new methods that can decrease the duration of bone transport (BT) distraction osteogenesis, we injected composite materials containing recombinant human bone morphogenetic protein‐2 (BMP‐2) and induced the generation of a callus bridge by rapid segmental transport (4 mm/day) in a rabbit bone defect model. The composite materials consisted of BMP‐2 (0, 30, or 100 µg), β‐tricalcium phosphate powder (βTCP, 100 mg/animal; particle size, <100 µm), and polyethylene glycol (PEG; 40 mg/animal). A paste of equivalent composition was percutaneously injected at the lengthening and the docking sites after surgery and after BT, respectively. The radiographic, mechanical, and histological examinations 12 weeks post‐operative revealed that the generation of bridging callus in the presence and in the absence of BMP‐2 was significantly different. The callus mass in the bone defect region was adequately and consistently developed in the presence of 100 µg of BMP (administered for 6 weeks), and the bones were consolidated in 12 weeks. Such an adequate callus formation was not observed in the control animals without BMP‐2 treatment. The result of this experimental study suggests the potential application of BMP‐2 in accelerating callus formation and in enabling rapid bone transporting, thereby shortening the treatment period for the repair of diaphyseal bone defects by distraction osteogenesis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:452–456, 2011     

Locally applied nerve growth factor enhances bone consolidation in a rabbit model of mandibular distraction osteogenesis.

Distraction osteogenesis is widely used in treating deformities, defects, and fractures of both long bones and craniofacial bones. Demands for acceleration of bone consolidation are increased in distraction osteogenesis. Nerve growth factor (NGF) can enhance innervation and bone regeneration in a fracture model and stimulate differentiation of osteoblastic cells. In this study, we tested the ability of locally applied NGF to enhance bone regeneration in a rabbit model of mandibular distraction osteogenesis. Twenty rabbits underwent bilateral distraction osteogenesis with a rate of 0.5 mm per 12 h. Two times 0.04 mg human NGFbeta (hNGFbeta) in buffer was injected into the callus after distraction. The contralateral side received placebo injections. Rabbits were euthanized at consolidation times of 14 and 28 days. Specimens were subjected to radiography, callus dimensions measurement, mechanical testing, and bone histological and histomorphometric analysis. The maximum load, bone volume/total volume, mineral apposition rate of the 1st to 11th day, and mineralized bone percentage were significantly higher in the hNGFbeta side at 14 and 28 days (p<0.05). The data indicate that locally applied hNGFbeta can accelerate callus maturation and may be an option to shorten the consolidation period in distraction osteogenesis.     

Effect of nerve growth factor and Schwann cells on axon regeneration of distracted inferior alveolar nerve following mandibular lengthening

To study the effect of nerve growth factor(NGF) and Schwann cells on axon regeneration of theinferior alveolar nerve following mandibular lengtheningwith distraction osteogenesis.     

Transplantation of osteoblast-like cells to the distracted callus in rabbits

We carried out limb lengthening in rabbits and then transplanted osteoblast-like cells derived from the tibial periosteum to the centres of distracted callus immediately after distraction had been terminated. Two weeks later the transaxial area ratio at the centre of the distracted callus and the bone mineral density (BMD) were significantly higher in the transplanted group, by 21% and 42%, respectively, than in the non-injected group or the group injected with physiological saline (p < 0.05). Callus BMD as a percentage of density in uninvolved bone was also significantly higher in the transplanted group (p < 0.05) than in the other two groups, by 27% and 20% in the second and fourth weeks, respectively (p < 0.05). Mechanically, the callus in the transplanted group tended to be stronger as shown by the three-point bending test although the difference in fracture strength was not statistically significant. Our results show that transplantation of osteoblast-like cells promotes maturity of the distracted callus as observed at the second and fourth weeks after lengthening. The method appears promising as a means of shortening the consolidation period of callus distraction and decreasing complications during limb lengthening with an external fixator.     

Callus formation in the humerus compared with the femur and tibia during limb lengthening

We investigated whether the callus formation in the humerus during the distraction period of limb lengthening proceeds at a higher rate than that in the femur and tibia. Ten achondroplastic patients underwent 3 bilateral humerus, 3 bilateral femur and 4 bilateral tibia lengthenings. To reduce the confounding effect of bone size, we used bone mineral apparent density (BMAD) to compare the three groups; this is a volumetric bone mineral density measurement. BMAD in the distracted callus space was evaluated at 8 weeks after the start of distraction using dual-energy X-ray absorptiometry (mean ± SD; g/cm³): in the humerus (0.24 ± 0.08) it was significantly higher than in the tibia (0.10 ± 0.02), while there was no difference between the humerus and femur (0.35 ± 0.11). We conclude that the callus formation in the humerus during the distraction period of limb lengthening proceeded at a significantly faster rate than in the tibia, but there was no significant difference between the humerus and femur. Received: 4 March 1997     

Stimulation of Bone Formation by Recombinant Fibroblast Growth Factor-2 in Callotasis Bone Lengthening of Rabbits

Bone lengthening by callotasis is one of the most useful methods not only for the treatment of short extremities but also for extensive bone defects; however, the procedure takes a long time especially for the consolidation of the distracted callus. In this study, effects of a single local injection of recombinant human fibroblast growth factor-2 (FGF-2 or basic FGF) on callotasis bone lengthening were examined in rabbits. Ten days after the osteotomy at the middle of the tibia and the installment of an external fixator, the osteotomized site was distracted at a rate of 1.4 mm/day for 7 days, resulting in 9.8 mm lengthening. On the final day of distraction, 200 μg of FGF-2 in 150 μl of saline solution or vehicle alone was injected into the center of the distracted callus. Injection of FGF-2 increased bone formation at the distracted callus radiologically and histologically. A significant effect on bone mineral content (BMC) at the callus was observed as early as 2 weeks, and FGF-2 increased the BMC about twofold at 5 weeks after a normal remodeling process. We conclude that the callotasis method in combination with FGF-2 injection at the consolidation step could be clinically beneficial to shorten the bone lengthening period. Received: 6 May 1997 / Accepted: 1 November 1998     

Dose-dependent effect of low-intensity pulsed ultrasound on callus formation during rapid distraction osteogenesis.

Distraction osteogenesis of bone or callotasis causes poor bone formation when the distraction rate is beyond the optimal rate. Low-intensity pulsed ultrasound was reported to enhance fracture healing, treatment of nonunion, and accelerate bone maturation and remodeling during consolidation stage of distraction osteogenesis. In this study, we evaluated the efficacy of different durations of LIPUS treatments during rapid bone lengthening. After 7-day latent period, osteotomized New Zealand white rabbit tibiae were lengthened at the rate of 2 mm per day for 1 week. Two different LIPUS treatment durations of 20 min and 40 min were selected for treatment groups. Rabbits without treatment served as the control group. Plain X-ray, peripheral quantitative computed tomography (pQCT) and histology were performed to assess bone acquisition in the distraction callus. The results showed that LIPUS increased bone mineral content and volume of the mineralized tissue of distraction callus in a dose-dependent manner. The different regions of distraction callus exhibited various spatial response to LIPUS treatment. Moreover, LIPUS enhanced dose-dependant endochondral formation. Compared with 20-min treatment, the 40-min LIPUS treatment was a more favorable treatment duration for bone regeneration in the distraction callus. In conclusion, LIPUS was able to enhance bone regeneration under rapid distraction, and its effect was dose-dependent.     

兔下颌骨牵拉成骨动物模型的建立及初步观察

目的　建立兔下颌骨牵拉成骨动物模型,研究了解颅面部牵拉骨生成的规律。方法　将12 只新西兰大白兔右侧下颌骨第一磨牙前完全截骨后用牵拉器固定,左侧不做手术为对照侧。1周后以每天一次0.9 m m 的速度逐步牵拉,连续10 天。牵拉完成后1 天,2、4 和8 周每组处死3只兔,取下完整下颌骨进行大体测量,X线摄片,新骨组织学观察。结果　12只兔右侧下颌骨平均延长8.3 m m ,与对照侧比较有显著性差异(P< 0.01),无骨不连及畸形愈合。X线摄片发现,延长完成后2周牵拉间隙已被骨痂桥接,8 周时,X线片上很难分辨新骨和正常骨。组织学观察牵拉早期即有胶原束形成,随后钙化成骨,未发现软骨中介体。结论　运用牵拉成骨技术可成功地延长兔下颌骨,无骨不连和畸形愈合等并发症,新骨以膜内成骨方式生成     

Local application of VEGF compensates callus deficiency after acute soft tissue trauma—results using a limb‐shortening distraction procedure in rabbit tibia

Acute soft tissue trauma influences callus formation and fracture healing. Several studies showed a relationship between angiogenesis and bone formation during distraction osteogenesis. The purpose of this study was to investigate the influence of controlled release of vascular endothelial growth factor (VEGF) on callus formation in a limb‐shortening distraction procedure after acute compartment syndrome. Acute soft tissue trauma with critical increased compartment pressure was generated in 22 rabbits, and the limb was shortened simulating fracture site debridement. In the test group (n = 11), a VEGF‐coated collagen matrix was locally applied around the fracture, while no collagen was applied in the control group (n = 11). Following 10 days in limb shortening, a gradual distraction of 0.5 mm/12 h was performed using an external fixation device and followed up for 40 days. Osseous consolidation occurred in all animals. Average callus diameter (1.54 ± 0.8 vs. 1.27 ± 0.14 mm) and torsional strength (72% vs. 46% of normal) were significantly higher in the test versus the control group. Blood vessel formation increased with a significantly higher number of vessels (6.3 vs. 3.81/mm²) and larger cross‐sectional area (>40 µm, 90.5% vs. 86%) in the test versus control group. The results showed that locally applied VEGF stimulates fracture healing after acute soft tissue trauma and might be an option for fracture treatment in cases with severe soft tissue damage. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1093–1098, 2011     

Histomorphometrical and radiological comparison of low-level laser therapy effects on distraction osteogenesis: experimental study

Distraction osteogenesis (DO) is the application of traction to the callus formed between bone segments and stimulation of bone formation by creating stress on the callus with this traction. Shorten the duration of DO and increasing the capacity of bone formation is important to prevent the possible complications of DO. For this reason, it was considered that low-level laser therapy (LLLT) may affect positively DO and it can decrease the complication range by shortening the period. Unilateral mandibular distractors were applied on 16 female white New Zealand rabbit to prove this hypothesis with micro CT, plain radiograph and histomorphometric analyses. Eight rabbits were applied LLLT with GaAlAs laser on the distraction area during the distraction period. On the post-distraction 28th day, four rabbits from study group and four rabbits from control groups were sacrificed. The rest of the rabbits were sacrificed on post-distraction 56th day. As a result of this study, significant positive effects of LLLT on post-distraction 28th day were revealed with all analyses. In histomorphometrical analyses, new bone formation was significantly higher in short-term laser applied group comparing to that of short-term control group (p?=?0.029). In both microCT and plain radiograph, the highest radioopacity values were observed in short-term laser group when compared with that of the controls (p?=?0.043 and p?=?0.025, respectively). Even though LLLT increased the healing capacity on short-term, it was not sufficient on long-term (post-distraction 56th day) healing. LLLT application on distraction period, activate healing on bone so it may decrease DO period. The result of this study should be supported with clinical studies and the most effective laser source, dose and application time should be revealed with experimental and clinical studies.     

Parathyroid hormone PTH(1�C34) increases the volume, mineral content, and mechanical properties of regenerated mineralizing tissue after distraction osteogenesis in rabbits

Background and purpose Parathyroid hormone (PTH) has attracted considerable interest as a bone anabolic agent. Recently, it has been suggested that PTH can also enhance bone repair after fracture and distraction osteogenesis. We analyzed bone density and strength of the newly regenerated mineralized tissue after intermittent treatment with PTH in rabbits, which undergo Haversian bone remodeling similar to that in humans.Methods 72 New Zealand White rabbits underwent tibial mid-diaphyseal osteotomy and the callus was distracted 1 mm/day for 10 days. The rabbits were divided into 3 groups, which received injections of PTH 25 µg/kg/day for 30 days, saline for 10 days and PTH 25 µg/kg/day for 20 days, or saline for 30 days. At the end of the study, the rabbits were killed and the bone density was evaluated with DEXA. The mechanical bone strength was determined by use of a 3-point bending test.Results In the 2 PTH-treated groups the regenerate callus ultimate load was 33% and 30% higher, absorbed energy was 100% and 65% higher, BMC was 61% and 60% higher, and callus tissue volume was 179% and 197% higher than for the control group.Interpretation We found that treatment with PTH during distraction osteogenesis resulted in substantially higher mineralized tissue volume, mineral content, and bending strength. This suggests that treatment with PTH may benefit new bone formation during distraction osteogenesis and could form a basis for clinical application of this therapy in humans.     

Fracture healing in protease‐activated receptor‐2 deficient mice

Protease‐activated receptor‐2 (PAR‐2) provides an important link between extracellular proteases and the cellular initiation of inflammatory responses. The effect of PAR‐2 on fracture healing is unknown. This study investigates the in vivo effect of PAR‐2 deletion on fracture healing by assessing differences between wild‐type (PAR‐2^+/+) and knock‐out (PAR‐2^?/?) mice. Unilateral mid‐shaft femur fractures were created in 34 PAR‐2^+/+ and 28 PAR‐2^?/? mice after intramedullary fixation. Histologic assessments were made at 1, 2, and 4 weeks post‐fracture (wpf), and radiographic (plain radiographs, micro‐computed tomography (µCT)) and biomechanical (torsion testing) assessments were made at 7 and 10 wpf. Both the fractured and un‐fractured contralateral femur specimens were evaluated. Polar moment of inertia (pMOI), tissue mineral density (TMD), bone volume fraction (BV/TV) were determined from µCT images, and callus diameter was determined from plain radiographs. Statistically significant differences in callus morphology as assessed by µCT were found between PAR‐2^?/? and PAR‐2^+/+ mice at both 7 and 10 wpf. However, no significant histologic, plain radiographic, or biomechanical differences were found between the genotypes. The loss of PAR‐2 was found to alter callus morphology as assessed by µCT but was not found to otherwise effect fracture healing in young mice. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1271–1276, 2012     

Effect of pulsed electromagnetic fields on maturation of regenerate bone in a rabbit limb lengthening model

To study the effect of applying pulsed electromagnetic fields (PEMF) during the consolidation phase of limb lengthening, a mid‐tibial osteotomy was performed in 18 adult New Zealand White rabbits and an external fixator was applied anteromedially. Animals were randomly assigned to treatment and control groups. After a 7‐day latency period, the tibiae were distracted 0.5 mm every 12 h for 10 days. The treatment group received a 20‐day course of PEMF for 60 min daily, coinciding with initiation of the consolidation phase. The control group received sham PEMF. Radiographs were performed weekly after distraction. Animals were euthanized 3 weeks after the end of distraction. Radiographic analysis revealed no significant difference in regenerate callus area between treatment and control tibiae immediately after distraction, at 1 week, 2 weeks, or 3 weeks after distraction ( p = 0.71, 0.22, 0.44, and 0.50, respectively). There was also no significant difference in percent callus mineralization ( p = 0.96, 0.69, 0.99, and 0.99, respectively). There was no significant difference between groups with respect to structural stiffness ( p = 0.80) or maximal torque to failure ( p = 0.62). However, there was a significant positive difference in mineral apposition rate between groups during the interval 1–2 weeks post‐distraction ( p < 0.05). This difference was no longer evident by the interval 2–3 weeks post‐distraction. While PEMF applied during the consolidation phase of limb lengthening did not appear to have a positive effect on bone regenerate, it increased osteoblastic activity in the cortical bone adjacent to the distraction site. Since the same PEMF signal was reported to be beneficial in the rabbit distraction osteogenesis when applied during distraction phase and consolidation phase, application of PEMF in the early phase may be more effective. Further work is necessary to determine optimal timing of the PEMF stimulation during distraction osteogenesis. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res