Viscoelastic characterization of mesenchymal gap tissue and consequences for tension accumulation during distraction

Nonlinear viscoelastic analysis was used to characterize the time-dependent behavior of mesenchymal gap tissue generated during distraction osteogenesis. Six (n = 6) lengthened tibiae were harvested from New Zealand white rabbits at 18 days. This gap tissue was subjected to a series of step displacement tests of increasing magnitude, and force relaxation behavior was monitored. Isochrones in stress-strain space were fit to odd cubic functions of strain. An analytic expression, linear in both e and e3, was developed to predict stress accumulation within the gap tissue as a function of time during distraction. Stress relaxation functions were described well by two-term Prony series. The two time constants determined from mechanical testing results were consistent, suggesting the presence of two fundamental physiologic relaxation processes. Gap tissue stresses were predicted to rise considerably during early stages of lengthening when distraction magnitudes exceeded the clinical norm of 0.25 mm. These differences in tension accumulation were less pronounced by the time lengthening was completed. Specifically, these results may in part explain clinical observations of decreased bone regeneration and altered tissue proliferation and differentiation at higher distraction rates. More generally, this work provides a framework for the rigorous characterization of the viscoelastic properties of biologic tissues ordinarily exposed to step strains.     

Characterization of midface maxillary membranous bone formation during distraction osteogenesis

The purpose of the study was to follow the early events in bone formation and neovascularization during maxillary distraction and after the consolidation period and to define the characterization of the new bone in the distracted area. Maxillary osteotomy was performed in seven sheep. In five animals, an external distraction device was used for maxillary lengthening of 20 mm at a rate of 1 mm/day for 20 days. Another two animals served as controls without distraction. Sequential biopsies were performed. The methods used for analysis were histologic, immunohistochemical, and ultrastructural by transmission electron microscopy. During the 5 days of latency, a fibrin clot was formed that after 5 days of distraction was replaced by granulation tissue, proliferating mesenchyme-like cells, and capillaries. After 10 days of distraction, the regenerated tissue could be divided into three main zones and two transitional areas: a central zone occupied by many polygonal mesenchyme-like cells and spindle-shaped cells that proliferated intensively; two paracentral zones on both sides of the central zone in which many cells showed morphologic signs of apoptosis leading to a decreased number of fibroblast-like cells embedded in wavy collagen fibers; a transitional area from the central to the paracentral zone in which concentric cellular colonies were believed to represent a novel form of vasculogenesis; distal-proximal zones, located on both sides of the paracentral zones and in continuation with the old bone, showed delicate new woven bone trabeculae that grew continuously in the direction of lengthening and gradually became mineralized; and a transitional area from the paracentral to the distal-proximal zones in which there was recruitment of preosteoblasts from the distracted tissue to the trabecular tips. These further differentiated into osteoblasts that contributed to the trabecular growth. The histologic feature pattern was similar after 15 and 20 days of continuous distraction. At the end of lengthening, after 20 days, delicate longitudinally oriented trabeculae continued to grow by recruiting preosteogenic cells from the central distracted tissue, became mineralized, and were rimmed by osteoblasts. After 6 weeks of retention, the trabeculae thickened and consisted of a mixture of lamellar and woven bone. In conclusion, the distraction force creates a pool of undifferentiated mesenchyme-like cells with osteogenic potential and triggers capillary formation, a clear zonation can be observed during active lengthening, and new bone trabeculae begin to form between 5 and 10 days after distraction, soon become aligned with osteoblasts, and continue to grow as long as distraction force is applied. This characterization may help in any exogenous involvement with growth factors to improve bone quality.     

Bone regeneration during distraction osteogenesis: mechano-regulation by shear strain and fluid velocity

Corroboration of mechano-regulation algorithms is difficult, partly because repeatable experimental outcomes under a controlled mechanical environment are necessary, but rarely available. In distraction osteogenesis (DO), a controlled displacement is used to regenerate large volumes of new bone, with predictable and reproducible outcomes, allowing to computationally study the potential mechanisms that stimulate bone formation. We hypothesized that mechano-regulation by octahedral shear strain and fluid velocity can predict the spatial and temporal tissue distributions seen during experimental DO. Variations in predicted tissue distributions due to alterations in distraction rate and frequency could then also be studied. An in vivo ovine tibia experiment evaluating bone-segment transport (distraction, 1 mm/day) over an intramedullary nail was used for comparison. A 2D axisymmetric finite element model, with a geometry originating from the experimental data, was created and included into a previously developed model of tissue differentiation. Cells migrated and proliferated into the callus, differentiating into fibroblasts, chondrocytes or osteoblasts, dependent on the biophysical stimuli. Matrix production was modelled with an osmotic swelling model to allow tissues to grow at individual rates. The temporal and spatial tissue distributions predicted by the computational model agreed well with those seen experimentally. In addition, it was observed that decreased distraction rate (0.5 mm/d vs. 0.25 mm/d) increased the overall time needed for complete bone regeneration, whereas increased distraction frequency (0.5 mm/12 h vs. 0.25 mm/6 h) stimulated faster bone regeneration, as found in experimental findings by others. Thus, the algorithm regulated by octahedral shear strain and fluid velocity was able to predict the bone regeneration patterns dependent on distraction rate and frequency during DO.     

High-impact exercise and growing bone: relation between high strain rates and enhanced bone formation.

We investigated whether high-impact drop jumps could increase bone formation in the middiaphyseal tarsometatarsus of growing rooster. Roosters were designated as sedentary controls (n = 10) or jumpers (n = 10). Jumpers performed 200 drop jumps per day for 3 wk. The mechanical milieu of the tarsometatarsus was quantified via in vivo strain gauges. Indexes of bone formation and mechanical parameters were determined in each of twelve 30 degrees sectors subdividing the middiaphyseal cortex. Compared with baseline walking, drop jumping produced large peak strain rates (+740%) in the presence of moderately increased peak strain magnitudes (+30%) and unaltered strain distributions. Bone formation rates were significantly increased by jump training at periosteal (+40%) and endocortical surfaces (+370%). Strain rate was significantly correlated with the specific sites of increased formation rates at endocortical but not at periosteal surfaces. Previously, treadmill running did not enhance bone growth in this model. Comparing the mechanical milieus produced by running and drop jumps revealed that jumping significantly elevated only peak strain rates. This further emphasized the sensitivity of immature bone to high strain rates.     

Spatial and temporal localization of WNT signaling proteins in a mouse model of distraction osteogenesis

While the surgical procedure of distraction osteogenesis (DO) is very successful in the treatment of orthopedic conditions, its major limitation of slow bone formation in the distracted gap has prompted numerous attempts to understand and accelerate this slow bone formation. Interestingly, WNT/FZD signaling has been identified as a critical pathway in mediating bone formation and regeneration but has not yet been studied in the context of DO. The objective of this study was to determine the spatial and temporal localization of endogenous WNT signaling proteins at various times of bone formation in a wild-type mouse model of DO. In this study, the DO protocol performed on mice consisted of three phases: latency (5 days), distraction (12 days), and consolidation (34 days). Our immunohistochemical findings of distracted bone specimens show an increased expression of WNT ligands (WNT4 and WNT10A), receptors (FZD1 and 2, LRP5 and 6), β-catenin, and pathway antagonizers (DKK1; CTBP1 and 2; sFRP1, 2, and 4) during the distraction phase, which were then down-regulated during consolidation. This is the first published report to show an activation of the WNT pathway in DO and could help identify WNT as a potential therapeutic target in accelerating bone regeneration during DO.     

Effect of distraction rate on biomechanical, mineralization, and histologic properties of an ovine mandible model

Craniofacial microsomia is a common congenital malformation. Ilizarov's method of distraction osteogenesis applied to the mandible has yielded promising results both experimentally and clinically. Because the technique is used predominantly in a pediatric population, length of treatment and compliance may be problematic. To date, the limits of distraction rate in the craniofacial skeleton have not been defined. This study was designed to investigate the effects of distraction rate, in a large animal model, on the mineralization, biomechanical, and histologic properties of lengthened mandibles. Clinically faster distraction rates would decrease the overall treatment time. Twenty-four animals were divided into four groups, with varying rates of distraction (1, 2, 3, and 4 mm/day). A uniaxial distractor at the angle of the mandible was used. The mandibles were lengthened to 24 mm and fixed for a period of 5 weeks, when the animals were killed. The specimens were analyzed with respect to mineralization using dual energy x-ray absorptiometry, biomechanical strength, through a modified three-point bending test, and histologic properties with hematoxylin and eosin stains. Biomechanical, mineralization, and histologic analyses of the samples indicated that group 1 (1 mm/day) samples were significantly superior (p<0.05) to those of group 4 (4 mm/day). Although bone formation was achieved in all groups, group 1 (1 mm/day) demonstrated the strongest biomechanical and histologic properties. Bone mineral density obtained using dual energy x-ray absorptiometry may be clinically useful as a reliable, noninvasive, and relatively cheap predictor for removal time of the fixator.     

Distraction osteogenesis of the porcine mandible: histomorphometric evaluation of bone

Distraction osteogenesis is a technique for skeletal lengthening that exploits the body's innate capacity for bone formation in response to tension forces on the repair callus. The authors developed a distraction osteogenesis model with a semiburied device in the Yucatan minipig mandible because of similarities between human and porcine mandibular anatomy, temporomandibular function, chewing patterns, and bone turnover rates. The purpose of this study was to measure histomorphometric bone fill after different latency periods, rates of distraction, and duration of neutral fixation in the minipig mandible. In addition, the relationship between histomorphometric bone fill and clinical stability was investigated. Mandibular osteotomies in 20 female Yucatan minipigs weighing 25 to 30 kg were distracted with modified semiburied distraction devices. Variables included 0-day or 4-day latency; 1-mm, 2-mm, or 4-mm daily distraction rates; gap size of 7 or 12 mm; and evaluation after neutral fixation for various lengths of time. Specimens were fixed in 2% paraformaldehyde, pH 7.4, before being embedded in methylmethacrylate. Sections were prepared from the region just below the inferior alveolar canal. The area of new bone formation within the gap was measured and expressed as a percentage of the total area of the distraction gap. Bone fill ranged from 0 to 100 percent. A pilot study with 7-mm advancements showed similar bone fill with 0-day or 4-day latency, but with poor reproducibility. Mandibles that were distracted to 12 mm at 1 mm per day exhibited nearly complete bone fill, either with 0-day latency (average, 93 percent) or 4-day latency (average, 100 percent). Mandibles that had been distracted for 3 days at 4 mm per day showed moderate osteogenesis and clinical stability with increasing time of neutral fixation. Bone fill was significantly correlated with clinical stability (Spearman r = 0.801, p = 0.001). Histological examination showed exuberant periosteal osteogenesis in distracted mandibles, even in those that showed poor bone fill and clinical stability. Thus, the periosteum appears to be a major source of new bone formation. These results show that osteogenesis was nearly complete with 1 mm per day and 0-day or 4-day latency. These results are consistent with the authors' previously reported clinical and radiographic observations that a latency period is not necessary for successful healing of the mandibular distraction osteogenesis wound.     

Three-dimensional histomorphometric analysis of distraction osteogenesis using an implanted device for mandibular lengthening in sheep

The aim of this study was to lengthen the sheep mandible with a fully buried device and to quantitatively analyze the tissue regenerate in the distraction gap by means of two-dimensional and three-dimensional histomorphometry. A custom-made device for continuous distraction was used in five adult sheep and fixed with three bicortical screws on either side of an osteotomy, anterior to the premolar region of the mandible. A cable-connected power and control unit was implanted in the neck region. After a 5-day latency period, distraction was activated every 2 hours and advanced at a rate of 1.01 mm per day. The distraction period was planned for 14 days, but because of stability problems and cable breakage, the actual distraction period ranged from 2 to 17 days, resulting in gap distances from 1.7 to 17.1 mm (mean, 0.95 mm/day). Osteogenesis was followed by radiographic imaging, and after a 6-week consolidation period, the harvested mandibles were serially sectioned for histologic and two-dimensional histomorphometric analysis, with three-dimensional reconstruction. Histologic examination of the specimens demonstrated predominantly membranous bone formation with remodeling bridging the distraction gap mainly in the periosteal region of the lingual side. In addition, cartilaginous areas and chondral bone formation were observed where the bridging appeared incomplete. Because of device fixation on the buccal side of the mandible, the preservation of the lingual periosteum seemed to play the major role for sufficient bone repair in the distraction gap. Cartilage within the distraction gap suggests fixation instability in this animal model.     

Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles.

We hypothesized that a 10-s rest interval (at zero load) inserted between each load cycle would increase the osteogenic effects of mechanical loading near previously identified thresholds for strain magnitude and cycle numbers. We tested our hypothesis by subjecting the right tibiae of female C57BL/6J mice (16 wk, n = 70) to exogenous mechanical loading within a peri-threshold physiological range of strain magnitudes and load cycle numbers using a noninvasive murine tibia loading device. Bone responses to mechanical loading were determined via dynamic histomorphometry. More specifically, we contrasted bone formation induced by cyclic vs. rest-inserted loading (10-s rest at zero load inserted between each load cycle) by first varying peak strains (1,000, 1,250, or 1,600 micro epsilon) at fixed cycle numbers (50 cycles/day, 3 days/wk for 3 wk) and then varying cycle numbers (10, 50, or 250 cycles/day) at a fixed strain magnitude (1,250 micro epsilon). Within the range of strain magnitudes tested, the slope of periosteal bone formation rate (p.BFR/BS) with increasing strain magnitudes was significantly increased by rest-inserted compared with cyclical loading. Within the range of load cycles tested, the slope of p.BFR/BS with increasing load cycles of rest-inserted loading was also significantly increased by rest-inserted compared with cyclical loading. In sum, the data of this study indicate that inserting a 10-s rest interval between each load cycle amplifies bone's response to mechanical loading, even within a peri-threshold range of strain magnitudes and cycle numbers.     

Diaphyseal bone formation in murine tibiae in response to knee loading.

Mechanical stimulation is critical for bone architecture and bone mass. The aim of this study was to examine the effects of mechanical loads applied to the knee. The specific question was whether loads applied to the tibial epiphysis would enhance bone formation in the tibial diaphysis. In C57/BL/6 mice, loads of 0.5 N were applied for 3 min per day for 3 days at 5, 10, or 15 Hz. Bone samples were harvested 13 days after the last loading. The strains were measured 13 +/- 2 microstrains at 5 Hz in the diaphysis. The histomorphometric data in the diaphysis clearly showed enhanced bone formation. First, compared with nonloaded control the cross-sectional cortical area was increased by 11% at 5 Hz and 8% at 10 Hz (both P < 0.05). Second, the cortical thickness was elevated by 12% at 5 Hz (P < 0.01) and 8% at 10 Hz (P < 0.05). Third, mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) were increased at 5 Hz (P < 0.01 for MS/BS; P < 0.001 for MAR and BFR/BS) and at 10 Hz (P < 0.05 for MS/BS; P < 0.01 for MAR and BFR/BS). Bone formation was enhanced more extensively in the medial side than the lateral or the posterior side. The results reveal that knee loading is an effective means to enhance bone formation in the tibial diaphysis in a loading-frequency dependent manner without inducing significant in situ strain at the site of bone formation.     

Local administration of allogeneic or autologous bone marrow-derived mesenchymal stromal cells enhances bone formation similarly in distraction osteogenesis

Background aimsDistraction osteogenesis (DO) is a surgical technique to promote bone regeneration that requires a long time for bone healing. Bone marrow-derived mesenchymal stromal cells (MSCs) have been applied to accelerate bone formation in DO. Allogeneic MSCs are attractive, as they could be ready to use in clinics. Whether allogeneic MSCs would have an effect similar to autologous MSCs with regard to promoting bone formation in DO is still unknown. This study compares the effect of autologous MSCs versus allogeneic MSCs on bone formation in a rat DO model.MethodsRat bone marrow-derived MSCs were isolated, characterized and expanded in vitro. Adult rats were subjected to right tibia transverse osteotomy. On the third day of distraction, each rat received one injection of phosphate-buffered saline (PBS), autologous MSCs or allogeneic MSCs at the distraction site. Tibiae were harvested after 28 days of consolidation for micro-computed tomography examination, mechanical test and histological analysis.ResultsResults showed that treatment with both allogeneic and autologous MSCs promoted bone formation, with significantly higher bone mass, mechanical properties and mineral apposition rate as well as expression of angiogenic and bone formation markers at the regeneration sites compared with the PBS-treated group. No statistical difference in bone formation was found between the allogeneic and autologous MSC treatment groups.ConclusionsThis study indicates that allogeneic and autologous MSCs have a similar effect on promoting bone consolidation in DO. MSCs from an allogeneic source could be used off-the-shelf with DO to achieve early bone healing.     

Mechanical characterization of regenerated osseous tissue during callotasis and its related biological phenomenon

The mechanical characteristics of the regenerated osseous tissue and osteoblastic activities during callotasis were studied using Chinese mountain goat as the animal model. Open osteotomy of the left tibiae was done in 24 goats. Distraction started 6 days after the operation with the rate of 1 mm per day for 4 weeks. The bone regeneration was monitored with serial X-ray films. The tension generated during distraction was measured with the strain gauges mounted on the distraction apparatus. The osteoblastic activities were monitored by measuring plasma bone specific alkaline phosphatase activity. The results showed that the average lengthening was 22.9 +/- 2.8 mm in each animal. The newly formed osseous tissue becomes stiffer during the course of distraction lengthening. The continuous evolution of the tensile behaviour of the newly formed osseous tissue correlates with the plasma bone specific alkaline phosphatase activities. The radiological appearance of a physis like structure took place at 12 mm lengthening. Its appearance corresponds to the changes in the tensile behaviour as well as the biological activities of the osteoblasts and may serve as a useful radiological marker in monitoring the process of callotasis in clinical practice.     

Transport distraction osteogenesis: a new method to heal adult calvarial defects

Popularized by Gavril Ilizarov in the 1960s, monofocal distraction osteogenesis has become a well-established method of endogenous bone engineering. This revolutionary surgical technique has significantly augmented the available reconstructive orthopedic and craniomaxillofacial procedures. Bifocal distraction osteogenesis, or bone transportation, is a modification of monofocal distraction that involves moving a free segment of living bone to fill an intercalary bone defect. Bifocal distraction has been applied successfully to reconstruct complex mandibular and long bone defects. Because traumatic or postsurgical calvarial defects do not spontaneously heal in humans older than 18 to 24 months of age, we hypothesized that bifocal distraction osteogenesis could be applied to the skull to close critical size calvarial defects. Critical size (15 x 15 mm) calvarial defects were created in eight New Zealand White rabbits. Next, a 15-mm x 10-mm calvarial box osteotomy was created just anterior to the skull defect. This osteotomy created a free bone segment that could be transported. A custom-made transport distraction device was fixed into place and the skin incision was closed. After a 4-day latency period, the distraction device was activated (0.5 mm once daily for 30 days) in seven animals; the distraction device in one animal was not activated and served as a control. All animals underwent 30 days of consolidation and were then killed. Radiographs and computed tomographic scans were performed at the following time points: end of latency period (postoperative day 4), mid-distraction (postoperative day 19), and end of consolidation period (postoperative day 64). Gross and histologic analysis was performed to evaluate the quality of the bony regenerate. The control animal healed with a fibrous union. Complete closure of the skull defects was observed in five of seven rabbits at the end of the consolidation period. One animal was removed from the study because of an early loosening of the distraction device, and one was removed because of device failure. Of the remaining five animals that completed the distraction protocol, radiographs and computerized tomographic scans showed successful ossification in all five rabbits at the end of the consolidation period. This study suggests that transport distraction osteogenesis is a promising technique that may be applied to a variety of commonly encountered craniofacial problems such as nonhealing calvarial defects.     

Low-magnitude mechanical signals that stimulate bone formation in the ovariectomized rat are dependent on the applied frequency but not on the strain magnitude

There is growing evidence that extremely small mechanical signals, if applied at a sufficiently high frequency, can serve as anabolic signals to bone tissue. To determine if the responsiveness of bone to low-magnitude, high-frequency parameters is modulated by endocrine imbalance, ovariectomized (OVX) Sprague-Dawley rats were subjected to whole body vibrations (WBV, 0.15 g) at 45 Hz (n=6) or 90 Hz (n=6) for 10 min/day, and compared to OVX age-matched controls (n=6). Five additional rats were used, in vivo, to establish the induced bone surface strain magnitudes (and strain rates). Following a 28 d protocol, bone formation rates in the metaphysis of the proximal tibia were 159% greater in 90 Hz rats when compared to age-matched controls, but 45 Hz rats were not significantly different from controls. Bone morphology of 90 Hz rats indicated significantly greater trabecular bone volume (22% and 25%) and thicker trabeculae (11% and 12%) over either controls or 45 Hz rats in the epiphysis of the distal femur, respectively. Despite the enhanced sensitivity of the skeleton towards the 90 Hz signal, the strain magnitudes and strain rates induced by this frequency were significantly lower than during 45 Hz vibration, suggesting that factors other than matrix strain are driving the anabolic response. Ideally, such mechanical signals represent a non-pharmacologic means of controlling bone mass and morphology in spite of systemic pressures for bone resorption.     

Reduced gap strains induce changes in bone regeneration during distraction.

A bilateral New Zealand white rabbit model of distraction osteogenesis (DO) was used to investigate the relationship between strain environment and bone regeneration during limb lengthening. In seven (n = 7) rabbits, a stiffener was applied to the fixator on one side to reduce strains within the gap tissue after lengthening was completed. Animals were euthanized six days later and their distraction zones were harvested and analyzed for changes in new bone volume and architecture. Nonlinear finite element analyses (FEA) were performed to predict changes in the gap strain environment. FEA results predicted a nearly uniform sevenfold decrease in average strain measures within the distraction zone. No change in total average new bone volume and significant decreases in both bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were observed in tibiae in which gap strains were reduced experimentally, compared to contralateral controls. These results suggest that fixator stiffening influenced the architecture but not the amount of newly formed bone. This animal model of distraction might be used to study the mechanisms by which strain fields affect events in bone repair and regeneration, such as cell proliferation, precursor tissue differentiation, and altered growth factor and nutrient delivery to tissues.     

Relationships between tissue dilatation and differentiation in distraction osteogenesis.

Mechanical factors modulate the morphogenesis and regeneration of mesenchymally derived tissues via processes mediated by the extracellular matrix (ECM). In distraction osteogenesis, large volumes of new bone are created through discrete applications of tensile displacement across an osteotomy gap. Although many studies have characterized the matrix, cellular and molecular biology of distraction osteogenesis, little is known about relationships between these biological phenomena and the local physical cues generated by distraction. Accordingly, the goal of this study was to characterize the local physical environment created within the osteotomy gap during long bone distraction osteogenesis. Using a computational approach, we quantified spatial and temporal profiles of three previously identified mechanical stimuli for tissue differentiation-pressure, tensile strain and fluid flow-as well as another candidate stimulus-tissue dilatation (volumetric strain). Whereas pressure and fluid velocity throughout the regenerate decayed to less than 31% of initial values within 20 min following distraction, tissue dilatation increased with time, reaching steady state values as high as 43% strain. This dilatation created large reductions and large gradients in cell and ECM densities. When combined with previous findings regarding the effects of strain and of cell and ECM densities on cell migration, proliferation and differentiation, these results indicate two mechanisms by which tissue dilatation may be a key stimulus for bone regeneration: (1) stretching of cells and (2) altering cell and ECM densities. These results are used to suggest experiments that can provide a more mechanistic understanding of the role of tissue dilatation in bone regeneration.     

Callus stimulation in distraction osteogenesis

Distraction osteogenesis has been described as in vivo tissue engineering. The ability to stimulate this process for the repair of bony defects or lengthening of congenitally shortened facial structures is likely to significantly impact the field of craniofacial surgery. The purpose of this study was to determine whether mechanical stimulation of the distracted rabbit mandible would accelerate the maturation of the bony callus when applied during the early consolidation period. Twenty adult New Zealand White rabbits underwent unilateral mandibular osteotomy. A uni-directional internal distractor device (Synthes, Paoli, Pa.) was positioned along a plane perpendicular to the line of osteotomy. After a 7-day latency period, distraction was commenced at a rate of 1.0 mm/day for 12 days in all animals. In a control group of 10 rabbits, a consolidation period of 8 weeks was observed before they were killed. In the experimental group of 10 rabbits, daily alternate compression and distraction of 1 mm (sequential compression and distraction) was performed for 3 weeks followed by a 5-week period of rigid fixation. Each animal received a dose of a fluorescent label at three different time points during the study: at the end of the distraction period, 3 weeks after the completion of the distraction phase, and 3 days before it was killed. All animals were killed 8 weeks after the completion of the distraction phase. Undecalcified histologic analysis and 3-point bending tests to failure were performed on the extracted mandibles. The results of the experimental and control groups were compared.Four animals in the control group and three animals in the experimental group were excluded from the study because of screw loosening resulting in distractor dislodgment or because of infection. On histologic analysis, cortical thickness at the center of the callus was found to be significantly greater in the experimental group compared with the control group when normalized to the contralateral hemimandible (83 percent versus 49 percent, respectively; p < 0.007). The ratio of cortical to cancellous bone in the distracted callus was uniformly found to be greater in the experimental specimens. The mineral apposition rate was calculated by using fluorescence microscopy and found to be significantly greater in the experimental group both during the period of sequential compression and distraction (3.2 microm/day versus 2.1 microm/day, p = 0.02) and after the period of sequential compression and distraction (1.4 microm/day versus 1.1 microm/day, p = 0.006). Mechanical testing revealed no significant differences in bending strength or stiffness between experimental or control groups (p = 0.54 and 0.47, respectively). This study has demonstrated that daily alternating compression and distraction of 1 mm amplitude during the early consolidation period has a stimulatory impact on callus formation with respect to osteoblastic activity, remodeling, and maturation of bone. Optimal timing and amplitude of sequential movement, long-term biomechanical differences, and molecular pathways have yet to be elucidated.     

Modeling distraction osteogenesis: analysis of the distraction rate

Distraction osteogenesis is a useful technique aimed at inducing bone formation in widespread clinical applications. One of the most important factors that conditions the success of bone regeneration is the distraction rate. Since the mechanical environment around the osteotomy site is one of the main factors that affects both quantity and quality of the regenerated bone, we have focused on analyzing how the distraction rate influences on the mechanical conditions and tissue regeneration. Therefore, the aim of the present work is to explore the potential of a mathematical algorithm to simulate clinically observed distraction rate related phenomena that occur during distraction osteogenesis. Improvements have been performed on a previous model (Gómez-Benito et al. in J Theor Biol 235:105–119, 2005) in order to take into account the load history. The results obtained concur with experimental findings: a slow distraction rate results in premature bony union, whereas a fast rate results in a fibrous union. Tension forces in the interfragmentary gap tissue have also been estimated and successfully compared with experimental measurements.     

Identification of apoptotic cell death in distraction osteogenesis

The purpose of this experimental work was to investigate whether apoptosis contributes to tissue remodelling during distraction bone healing. In a rabbit model of mandibular distraction osteogenesis, we quantitatively analysed the extent of apoptotic cell death in relation to differently applied mechanical loadings. Apoptotic cells were identified by means of an in situ detection assay for nuclear DNA fragmentation using a modified TUNEL procedure and by electron microscopical examination for typical morphological features of programmed cell death. TUNEL-positive cells were frequently detected in samples distracted at higher strain magnitudes. Ultrastructurally, these apoptotic cells displayed a condensed chromatin and fragmented nuclei, while the continuity of their plasma membranes remained intact. Our results clearly indicated that the discontinuous traction of osteotomized mandibles induced enhanced apoptosis. In contrast to non-distracted samples and mandibles distracted at low strain magnitudes, in which only minimal evidence of apoptotic cell death was detected, the application of hyperphysiological strain magnitudes resulted in an increased apoptosis rate. Thus, mechanical loading seems to be a triggering factor for apoptotic changes in osteoblastic cells. These findings suggest a pathophysiological role of apoptotic cell death in the control of tissue integrity during distraction osteogenesis.