Inhibin A enhances bone formation during distraction osteogenesis

Given the aging population and the increased incidence of fracture in the elderly population, the need exists for agents that can enhance bone healing, particularly in situations of delayed fracture healing and/or non‐union. Our previous studies demonstrated that overexpression of the gonadal peptide, human inhibin A (hInhA), in transgenic mice enhances bone formation and strength via increased osteoblast activity. We tested the hypothesis that hInhA can also exert anabolic effects in a murine model of distraction osteogenesis (DO), using both transgenic hInhA overexpression and administration of normal physiological levels of hInhA in adult male Swiss‐Webster mice. Tibial osteotomies and external ring fixation were performed, followed by a 3‐day latency period, 14‐day distraction, and sacrifice on day 18. Supraphysiological levels of hInhA in transgenic mice, but not normal physiological levels of hInhA, significantly increased endosteal bone formation and mineralized bone area in the distraction gap, as determined by radiographic and µCT analysis. Significantly, increased PCNA and osteocalcin expression in the primary matrix front suggested that hInhA increased osteoblast proliferation. This mechanism is consistent with the effects of other agents and pathologies that modulate bone formation during DO, and demonstrates the potential of hInhA to enhance bone repair and regeneration. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:288–295, 2012     

Effect of weightbearing on bone formation during distraction osteogenesis

The effect of weightbearing on the bone formation during distraction osteogenesis was studied with 24 goats after standardized osteotomy and distraction. Twelve goats were allowed to bear weight immediately and 12 were not allowed to bear weight. With the pattern of weightbearing documented, the formation of bone was monitored with serial radiographs and the bone forming activity was studied with histologic examination and immunohistochemical study of transforming growth factor-beta 1 expression. The results of the study showed that the bone formation as depicted by radiologic assessment of callus width and mineralized tissue was significantly better in the weightbearing group. Histologic study showed more bone formation qualitatively and quantitatively in the weightbearing group. This study confirmed the beneficial effect of weightbearing activity during distraction osteogenesis. Distraction provides pulsed form stimulation by tension stress across the osteotomy site and initiates osteogenesis while the compression stress exerted by the weightbearing is continuous in daily activities and enhances new bone formation. The clinical practice of early weightbearing during distraction osteogenesis should be reinforced.     

Radiographic assessment of bone formation in tibia during distraction osteogenesis

The radiologic appearance of bone formation during limb lengthening is used to judge the competence of the underlying biologic process and predict the likely time to healing. Interpretation is, however, based upon subjective parameters that have never been clearly defined. Thirty anteroposterior radiographs from pediatric tibial lengthenings were classified by four pairs of surgeons using a three-part system. Across the group, interobserver consistency showed considerable variation for all parameters tested. Pairwise analysis indicated that the surgeons directly involved in limb lengthening procedures agreed better than those whose practice was of a different nature. A second series of radiographs contained 12 radiographs with a satisfactory and 12 with a poor bone healing index (BHI). These radiographs were digitized, and an interobserver study showed significantly improved observer concordance if the images were enhanced. No feature was associated with a 100% chance of a satisfactory outcome, but certain appearances in the regenerate were associated with a better BHI than others.     

Effect of alpha-tocopherol on bone formation during distraction osteogenesis: a rabbit model

Purpose  

The purpose of this study was to evaluate the effects of alpha-tocopherol on distraction osteogenesis.     

Effects of rapid distraction rate on new bone formation during mandibular distraction osteogenesis in goats

Objective

Distraction osteogenesis typically requires a long treatment period, which can lead to bone and soft-tissue infection and considerable patient discomfort. Use of a rapid distraction rate in craniofacial distraction osteogenesis to shorten the distraction period is possible owing to the unique characteristics of craniofacial bones, including an abundant blood supply and rapid bone healing compared with long bones. The effects of using a rapid distraction rate in the treatment of craniofacial deformities are currently unclear, however. The objective of this study was to investigate the effects of a rapid distraction rate on new bone formation during mandibular distraction osteogenesis in goats.

Methods

Sixteen goats were randomly divided into four groups consisting of four goats each. In Groups A, B, and C, the right mandible of each goat was distracted at a rate of 0.8 mm/d, 1.6 mm/d, and 2.0 mm/d, respectively; Group D was the control group and did not undergo distraction. Six weeks after the conclusion of distraction, bone densitometry and three-point bending testing were performed in all groups.

Results

The mean bone density value of goats in Group A was significantly higher than those of all the other groups (p < 0.05), and the mean bone density value of goats in Group C was significantly lower than those of all the other groups (p < 0.05). The mean curve slope, peak stress, bending modulus, and energy to failure values of Groups A, B, and C were all significantly lower than those of the control group (p < 0.05). As the distraction rate increased, the curve slope and peak stress values gradually declined (p < 0.05).

Conclusions

Use of a rapid distraction rate in mandibular distraction osteogenesis may have detrimental effects on the quality of new bone, despite the abundant blood supply of craniofacial bones.     

Cisplatin inhibits bone healing during distraction osteogenesis

Osteosarcoma (OS) is the most common malignant bone tumor affecting children and adolescents. Many patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. Surgical reconstructions after tumor resection include structural allografts, non‐cemented endoprostheses, and distraction osteogenesis (DO), which require direct bone formation. Although cisplatin (CDP) is extensively used for OS chemotherapy, the effects on bone regeneration are not well studied. The effects of CDP on direct bone formation in DO were compared using two dosing regimens and both C57BL/6 (B6) and tumor necrosis factor receptor 1 knockout (TNFR1KO) mice, as CDP toxicity is associated with elevated TNF levels. Detailed evaluation of the five‐dose CDP regimen (2 mg/kg/day), demonstrated significant decreases in new bone formation in the DO gaps of CDP treated versus vehicle treated mice (p < 0.001). Further, no significant inhibitory effects from the five‐dose CDP regimen were observed in TNFR1KO mice. The two‐dose regimen significantly inhibited new bone formation in B6 mice. These results demonstrate that CDP has profound short term negative effects on the process of bone repair in DO. These data provide the mechanistic basis for modeling peri‐operative chemotherapy doses and schedules and may provide new opportunities to identify molecules that spare normal cells from the inhibitory effects of CDP. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:464–470, 2014.     

Rosiglitazone disrupts endosteal bone formation during distraction osteogenesis by local adipocytic infiltration

Rosiglitazone (Rosi) is a drug in the thiazolidinedione class for treatment of type 2 diabetes mellitus (T2DM), which binds and activates PPARγ nuclear receptor in fat cells, sensitizing them to insulin. Despite proven antidiabetic efficacy, Rosi therapy may be associated with trabecular bone loss and an increased risk of fractures. To examine the potential side effects of Rosi treatment on bone formation, we delivered Rosi to mice using a combined model of distraction osteogenesis (DO) and type 2 diabetes mellitus (T2DM). DO provides a unique method to isolate the sequence of intramembranous bone formation, an important component of both fracture healing and bone homeostasis. Four groups of n = 6 mice were used to compare the effects of Rosi on bone formation and cellular composition in both diabetic (Avy/a strain) and non-diabetic mice (a/a strain). New bone formation was examined by high resolution radiographs, micro-computed tomography, and histology. Precursor cells in the distraction gap were quantitated using immunohistochemical stains for proliferating cell nuclear antigen. Committed osteoblasts and adipocytes in the gap were identified and quantitated by immunostaining for osteocalcin and FABP4/aP2, respectively. The diabetic model developed obesity, hyperglycemia, hyperinsulinemia and insulin resistance, while the control littermates remained lean, normoglycemic and insulin sensitive. Rosi treatment decreased levels of non-fasted glucose and insulin and improved insulin sensitivity in the A^vy/a mice, but had no effect in a/a mice, indicating antidiabetic efficacy of Rosi at the tested dose. Despite the diabetic, obese mice having twice the number of fat cells in their marrow than the non-diabetic mice, bone formation using DO was not adversely affected by the diabetes itself. However, Rosi treatment significantly diminished intramembranous endosteal bone formation, while increasing adipogenesis in and adjacent to the distraction gap up to 3.5- to 3.8-fold in both diabetic and non-diabetic models. This effect was independent of the anti-diabetic therapeutic response. These results raise the question of whether osteoblast precursors are inhibited in their development or actually converted to adipocytic phenotypes, possibly via marrow fat PPARγ nuclear receptor.     

Radiographic classification of osteogenesis during bone distraction.

Successful limb lengthening requires serial radiological evaluation of the progression of healing of the regenerate bone. However, there is no radiographic classification system that shows how the regenerate should progress during treatment in adults. The study aimed to address this need.A series of radiographs were studied from 92 patients (125 segments) who had undergone bone lengthening. A radiographic classification of osteogenesis was developed based on callus shape and radiographic features that occur between osteotomy and fixator removal.This classification system used both shape and type of feature to condense and record the radiographic information, but type of feature alone was sufficient to predict outcome. The concurrence and reproducibility of the classification system was tested by inter- and intra-observer studies. The degree of consistent repetition and agreement between observers suggests that the classification system is reliable, reproducible, and therefore should be robust in use.This classification system provides an insight into osteogenesis; it allows the progress of the bone healing to be assessed against a successful pattern of healing. Hence, potential problems can be predicted and clinical changes made to improve outcome. The classification can be simplified to make it more appropriate for clinical use.     

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.     

Monitoring of bone formation during distraction osteogenesis via osteocalcin: a time sequence study in dogs

 The aim of the current study was to analyze the osteocalcin level and radiographic density during distraction osteogenesis in order to investigate the role of osteocalcin in monitoring bone formation during callus distraction. Lengthening of the right tibia by 25% was performed in 12 beagle dogs by callus distraction after osteotomy and application of a ring fixator. Distraction was started on the 5th postoperative day, with a distraction rate of 0.5 mm twice a day, and was ended after 25 days. Blood samples and x-rays of the callus distraction segment were obtained preoperatively and once a week until day 55 after operation. A digital radiograph analysis system was used to determine the bone density of the callus distraction segments. The serum parameters of osteocalcin were evaluated by radioimmunoassay. The radiographic bone densities during the distraction phase increased during the distraction period and markedly increased during the consolidation period. A similar trend was observed for osteocalcin, whereby the coefficient of correlation between these two parameters was, on average, 0.68 ± 0.11. However, the radiographic bone density measurements, as well as the osteocalcin levels, showed large variation between different animals. Therefore, our results suggest that valuable information about bone formation during distraction osteogenesis can be obtained via serum osteocalcin levels, even though it seems that time sequence monitoring is more favorable than the determination of absolute values. Received: January 16, 2002 / Accepted: April 10, 2002     

Enhancing bone healing during distraction osteogenesis with platelet-rich plasma

Gradual limb lengthening with external fixators using distraction osteogenesis principles is the gold standard for treatment of limb-length discrepancy. However, long treatment time is a major disadvantage of the current lengthening procedures. Efforts to decrease the treatment include biological and biomechanical factors. Injection of platelet-rich plasma (PRP) is a biological method to enhance bone healing during distraction osteogenesis. We hypothesised that PRP can enhance bone healing during limb lengthening. We report our experience with the use of PRP during distraction osteogenesis. This retrospective study included 19 patients divided into the standard group of 10 patients who did not receive PRP and the PRP group of nine patients who received PRP at the end of the distraction phase. The study variables included external fixator time, external fixation index, and complications during treatment. The PRP group had statistically significantly shorter treatment time (p = 0.0412). Injection of PRP into regenerate bone might be an effective method to shorten treatment time during limb lengthening and lead to better functional outcomes and improved patient satisfaction.Level of evidence: Level IV, therapeutic study.     

Assessment of cell proliferation in regenerating bone during distraction osteogenesis at different distraction rates

An experimental model of lengthening of the lower limb was used to study the morphology and cellular proliferation of regenerating bone tissue after 20% lengthening at four rates of distraction. Groups of rabbits were killed at different times 1-8 weeks after surgery. The regenerated area was divided into three zones: fibrous, primary mineralization front, and new bone. As the rate of distraction increased, the size of the fibrous zone increased and that of Ihe new bone. one decreased. Necrosis, formation of cysts, and cartilage were found in the regenerated area at the higher distraction rates. Cell proliferation was assessed by in vivo labelling with' bromodeoxyuridine and the positive staining index for anti-bromodeoxyuridine antibody was calculated in the zones of the regenerated tissue, The index values for the fibrous zones and the new bone zones did not differ significantly in any of Ihe groups, The value increased significantly (p < 0,05) in the primary mineralization front as the rate of distraction increased from 0.3 to 0.7 mm/day, but there was no further significant increase at higher distraction rates. In conclusion, cell proliferation was increased at all of the higher rates (more than 0.3 mm/day) of distraction studied. Higher rates of distraction caused tissue damage. A distraction rate of 0.7 mm/day appeared optimal for cell proliferation and histological characteristics.     

Sclerostin antibody enhances bone formation in a rat model of distraction osteogenesis

Neutralizing monoclonal sclerostin antibodies are effective in promoting bone formation at a systemic level and in orthopedic scenarios including closed fracture repair. In this study we examined the effects of sclerostin antibody (Scl‐Ab) treatment on regenerate volume, density, and strength in a rat model of distraction osteogenesis. Surgical osteotomy was performed on 179 Sprague Dawley rats. After 1 week, rats underwent distraction for 2 weeks, followed by 6 weeks for consolidation. Two treatment groups received biweekly subcutaneous Scl‐AbIII (a rodent form of Scl‐Ab; 25 mg/kg), either from the start of distraction onward or restricted to the consolidation phase. These groups were compared to controls receiving saline. Measurement modalities included longitudinal DXA, ex vivo QCT, and microCT, tissue histology, and biomechanical four‐point bending tests. Bone volume was increased in both Scl‐Ab treatments regimens by the end of consolidation (+26–38%, p < 0.05), as assessed by microCT. This was associated with increased mineral apposition. Importantly, Scl‐Ab led to increased strength in united bones, and this reached statistical significance in animals receiving Scl‐Ab during consolidation only (+177%, p < 0.01, maximum load to failure). These data demonstrate that Scl‐Ab treatment increases bone formation, leading to regenerates with higher bone volume and improved strength. Our data also suggest that the optimal effects of Scl‐Ab treatment are achieved in the latter stages of distraction osteogenesis. These findings support further investigation into the potential clinical application of sclerostin antibody to augment bone distraction, such as limb lengthening, particularly in the prevention of refracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1106–1113, 2018.

     

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.     

Vascular tissues are a primary source of BMP2 expression during bone formation induced by distraction osteogenesis

Prior studies showed that bone regeneration during distraction osteogenesis (DO) was dependent on vascular tissue development and that inhibition of VEGFR signaling diminished the expression of BMP2. A combination of micro-computed tomography (μCT) analysis of vascular and skeletal tissues, immunohistological and histological analysis of transgenic mice containing a BAC transgene in which β-galactosidase had been inserted into the coding region of BMP2 and qRT-PCR analysis, was used to examine how the spatial temporal expression of the morphogenetic signals that drive skeletal and vascular tissue development is coordinated during DO. These results showed that BMP2 expression was induced in smooth muscle and vascular endothelial cells of arteries and veins, capillary endothelial cells, hypertrophic chondrocytes and osteocytes. BMP2 was not expressed by lymphatic vessels or macrophages. Separate peaks of BMP2 mRNA expression were induced in the surrounding muscular tissues and the distraction gap and corresponded first with large vessel collateralization and arteriole remodeling followed by periods of angiogenesis in the gap region. Immunohistological and qRT-PCR analysis of VEGF receptors and ligands showed that mesenchymal cells, lining cells and chondrocytes, expressed VEGFA, although PlGF expression was only seen in mesenchymal cells within the gap region. On the other hand VEGFR2 appeared to be predominantly expressed by vascular endothelial and hematopoietic cells. These results suggest that bone and vascular tissue formation is coordinated via a mutually supporting set of paracrine loops in which blood vessels primarily synthesize the morphogens that promote bone formation while mesenchymal cells primarily synthesize the morphogens that promote vascular tissue formation.     

Denervation impairs bone regeneration during distraction osteogenesis in rabbit tibia lengthening

Background and purposes The nervous system plays an important role in bone metabolism. However, the effect of denervation on bone formation during distraction osteogenesis (DO) remains unclear. We studied neural influence on bone regeneration during DO in a rabbit model. Methods 24 New Zealand male white rabbits underwent left tibial osteodistraction. Before distraction, the animals were randomly divided into group R (resected left sciatic nerve) and group I (intact left sciatic nerve). 8 weeks after completion of distraction, the animals were killed and the lengthened tibias were harvested for radiography, micro-CT, histological evaluation, and mechanical testing. Results New regenerated bone was present in the distraction gaps of all animals at the end of the study, as revealed by radiography, micro-CT, and histology. However, less new bone formation and a lower degree of mineralization were observed in group R. The mechanical strength of the distraction gap in group I was 1.3-fold greater than that in group R when measured using the 3-point bending test. Interpretation The results suggest that the nervous system plays an essential role during DO: the denervation appears to have an inhibitory effect on bone formation.     

生物因子促进牵张成骨区骨生成与矿化的研究进展

牵张成骨(distraction osteogenesis,DO)是一种利用骨痂愈合机制产生新骨的内源性组织工程学技术。随着近几年大量基础研究和临床应用的深入,牵张成骨在矫正先天性肢体不等长、创伤后遗症,创伤性骨髓炎、肿瘤术后重建等方面展现了广阔的应用前景。如何促进牵张成骨区骨生成与矿化、缩短患者疾病的治疗周期已成为国内外矫形外科领域研究的热点之一,本文就生物因子促进骨生成与矿化的应用进行综述。     

Mid-tibial distraction osteogenesis redistributes bone blood flow

Background Distraction osteogenesis can be used for the treatment of osteomyelitis and nonunion, conditions thought to benefit from increased blood flow in the bone tissue of the distracted limb. We have questioned whether such an increase occurs, and investigated the spatial distribution of bone blood flow after distraction osteogenesis.

Methods The tibiae of 8 rabbits were lengthened 10 mm by a standard midtibial distraction osteogenesis procedure. 2 weeks into the consolidation phase, the bone and soft tissue blood flow of the distracted and the contralateral extremity were measured using radioactive microspheres.

Results The absolute bone blood flow of the distracted tibia was 4% lower than that of the non-distracted side, representing a 41% decrease in the proximal metaphysis, a smaller decrease in the proximal epiphysis, distal metaphysis and distal epiphysis, and an increase in the diaphysis.

Interpretation Mid-tibial distraction osteogenesis redistributed the bone blood flow of the distracted tibia, but absolute tibial blood flow did not increase. Our results do not confirm previous research in this field.