Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study.

The presence of osteogenic progenitors in human skeletal muscle is suggested by the formation of ectopic bone in clinical and experimental conditions, but their direct identification has not yet been demonstrated. The aims of this study were to identify osteogenic progenitor cells in human skeletal muscle tissue and to expand and characterize them in culture. Specimens of gracilis and semitendinosus muscle were obtained from young adults and digested to separate the connective tissue and satellite cell fractions. The cells were cultured and characterized morphologically and immunohistochemically using antibodies known to be reactive with primitive osteoprogenitor cells, pericytes, intermediate filaments, and endothelial cells. Alkaline phosphatase activity and osteocalcin gene expression were also determined. In the early stages of culture, the connective tissue cells obtained were highly positive for primitive osteoprogenitor cell and for pericyte markers. Alkaline phosphatase activity was detectable at early stages of culture and rose as a function of time, whereas primitive osteoprogenitor cell markers declined and osteocalcin mRNA expression became detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). It is shown that human skeletal muscle connective tissue contains osteogenic progenitor cells. Their identification as pericytes, perivascular cells with established osteogenic potential, suggests a cellular link between angiogenesis and bone formation in muscle tissue. These cells are easily cultured and expanded in vitro by standard techniques, providing an alternative source of osteogenic progenitor cells for possible cell-based therapeutic use in certain conditions.

Muscle fibre damage and regeneration resulting from surgical limb distraction.

Using an animal model of limb distraction, the extent of muscle fibre damage and atrophy resulting from distraction at two different rates (1.3 or 3.0 mm day(-1)) was investigated. It was found that at the high rate of distraction there was a significantly greater loss of range of joint movement and more muscle fibre atrophy and fibre damage than at the low rate. Muscle fibre damage is usually followed by regeneration. This involves the expression of the neonatal form of myosin heavy chain, which can therefore be used as an indicator of regeneration. It was found that whilst many more fibres showed evidence of damage at the high compared to the low rate, the number of fibres expressing neonatal myosin was significantly reduced, indicating the presence of a population of fibres which was undergoing degeneration without subsequent regeneration. Thus it would appear that beyond a certain rate of distraction, regeneration may be insufficient to replace contractile material damaged by overstretching. It is suggested that these fibres are replaced with connective tissue. This process may contribute to the muscle weakness and loss of range of joint movement which sometimes accompanies limb distraction procedures.

Rapid application fracture fixators - an evaluation of mechanical performance.

OBJECTIVE: This study evaluates the mechanical performance of the Pinless and Centrafix fixators for rapid application to tibial fractures in a disaster or battlefield scenario. DESIGN: Comparative study based on measurements made in the laboratory. BACKGROUND: The Pinless and Centrafix fixators may be considered for rapid application to stabilise fractures in emergency conditions without the aid of electrical equipment such as power drills for bone screw insertion or image intensifiers to facilitate bone alignment. METHODS: Stiffnesses, maximum service loads and fatigue strengths of the fixators were measured in the orientations of loading that correspond to walking and stretcher-bearing. These properties were compared with measurements on three conventional fixators, the AO, Shearer and Triax. RESULTS: The Centrafix stiffnesses were 31 N/mm (axial), 1 N/ degrees (torsional shear), 0.4 N/ degrees (coronal plane bending), 4 N/ degrees (sagittal plane bending) and 11 N/mm (transverse shear) and strengths were 95 N (axial) and 1.9 Nm (bending). Corresponding Pinless stiffnesses were 43 N/mm, 0.7 N/ degrees, 0.3 N/ degrees, 8 N/ degrees and 50 N/mm, and strength was 55 N (axial). CONCLUSIONS: The stiffness and strength of both rapid application fixators in simulated walking was judged to be low, and additionally the stiffness and strength of the Centrafix in simulated stretcher-bearing was judged to be low. RELEVANCE: The Pinless is not recommended for weight-bearing subjects with unstable fractures. The Centrafix is not recommended for stretcher-bearing or weight-bearing with unstable fractures.

Fracture after distraction osteogenesis.

We reviewed 173 patients undergoing distraction osteogenesis to determine the incidence, location and timing of fractures occurring as a complication of the procedure. There were 17 fractures in 180 lengthened segments giving an overall rate of fracture of 9.4%. Unexpectedly, the pattern and location of the fractures were very variable; six were within the regenerate itself, six at the junction between the regenerate and the original bone and five at distant sites in the limb. Of those occurring in the regenerate, five were noted to be associated with compression and partial collapse of the regenerate. In three patients collapse and deformity developed gradually in the distracted segment over the six months after removal of the frame. The method of treatment of these fractures should be chosen to take into account multiple factors, which are additional and often different from those to be considered during management of acute traumatic injuries. Internal fixation appears to be most appropriate for displaced fractures, although in small children, or in those in whom there has been, or is, infection of the screw tracks, a new period of treatment using external fixation may be needed. Fixation by intramedullary nailing was associated with a risk of infection, even if screw tracks were assessed as healthy at the time of insertion of the nail. Internal fixation with the use of plates is safe for displaced, unstable fractures in children.

Correction of complex lower limb deformities by the Ilizarov technique: An audit of complications.

This is an audit of complications resulting from correction of complex lower limb deformities by the Ilizarov technique. 33 patients (40 bone segments) were reviewed and divided into 4 groups according to the type of surgery carried out: limb lengthening and/or correction of deformity, bone or joint stabilisation, treatment of nonunion or bone defect, angular and/ or rotation correction. Most minor complications were fixator specific. Pin tract infections were almost universal but responded well to oral antibiotics and rarely resulted in osteomyelitis. Major complications were procedure specific and more common in those patients who underwent leg lengthening, treatment for nonunion and bone transport. There was also a high incidence of nerve injury as a result of acute angular deformity correction. Despite the high complication rate the Ilizarov technique remains an effective tool for complex lower limb reconstruction surgery.

Stiffness, strength and healing assessment in different bone fractures--a simple mathematical model.

External fixators can only be removed safely when fractures have healed sufficiently to restore mechanical integrity to the bone. A bending stiffness of 15 N m/ degrees has been suggested as a means of estimating mechanical integrity. To examine whether this end point stiffness value can be applied to all fractures, the present study examined the degree of variability in predicted stiffness and strength that arises from variations in bone dimensions. Results imply that there is no common value for the end-point of bending stiffness in different bones. At an end point value of 15 N m/degrees, the maturity of the fracture repair tissue (represented by its elastic modulus) can vary 500-fold between an adult femur with a 0.5-mm gap to a child's mid diaphyseal tibia with a 1.0-mm gap. Fortunately, the strength does not vary by as large an extent as the modulus. However, even though two fractures each have reached a stiffness of 15 N m/degrees, a fracture in a bone of 50 mm diameter may exhibit only 60% of the strength of repair in a bone of 30 mm diameter. Therefore, caution should be exercised when using the bending stiffness as an end point indicator for different bones.

Effect of lengthening rate on angiogenesis during distraction osteogenesis.

This study investigated the angiogenic response to four varying rates (0.3, 0.7, 1.3, and 2.7 mm/day) of distraction in a rabbit model of leg-lengthening. Immunostaining was performed with use of specific antibodies to type-IV collagen and endothelial cell antigen to examine semiquantitatively the presence of blood vessels in the developing tissues. With use of the Chalkley counting method, the greatest number of positive-staining blood vessel cells was found in the central fibrous zone of the groups that underwent lengthening at 0.7 and 1.3 mm/day compared with any other zone in any group (p < 0.05, t test). There were no statistical differences in the positive labeling indices in the mineralization front and the new bone zone adjacent to the mineralization front in any of the groups. However, the decrease in the number of positive-staining blood vessel cells in the new bone zone distant to the mineralization front compared with any other zone in any group was statistically significant. The results suggest that during distraction osteogenesis, the precursor cells of new capillaries were present in abundance within the fibrous interzone. These cells connected into the capillary network at the junction of the mineralization front and the fibrous zone. The angiogenic response was weaker in the more mature regions within the new bone zones. A slow rate of distraction (0.3 mm/day) did not maximally stimulate angiogenesis in the central fibrous zone, whereas high rates (2.7 mm/day) appeared to impair this response. In this model of distraction osteogenesis, the vascularization process in the central fibrous zone was maximally stimulated at distraction rates of 0.7 and 1.3 mm/day.

Effect of rate of distraction on loss of range of joint movement, muscle stiffness, and intramuscular connective tissue content during surgical limb-lengthening: a study in the rabbit.

Surgical lengthening of limbs often results in loss of range of joint movement and this has been shown to be associated with an increase in passive tension and an increase in collagen content of the muscles. In this study, we have investigated the length/tension properties and the connective tissue component of muscle distracted at three different rates in order to determine whether low rates of distraction would enable the connective tissue component, as well as the contractile component (number of serial sarcomeres), to adapt more completely to the increased functional length of the muscle and thus lead to improved range of joint movement. It was found that loss of range of movement varied with rate of distraction. At the low rate, there was no change in the passive tension or collagen content compared to muscles from sham-operated animals, and range of movement was significantly greater than at the other rates. At the medium rate, although the muscles showed good adaptation in terms of serial sarcomere number, passive tension and collagen content was increased and range of movement reduced, indicating that changes in the connective tissue component are important factors in loss of joint movement. In the case of muscle distracted at a high rate, failure of the muscle fibres to add on sufficient sarcomeres, combined with changes in the connective tissue, resulted in almost total loss of joint movement.

Biologic model of bone transport distraction osteogenesis and vascular response.

We developed an experimental model in the rabbit of distraction osteogenesis through bone transport that closely corresponds to the clinical use of bone transport in humans. We also applied injection angiography to study the arterial response of a limb undergoing bone transport. This model includes a proximal osteotomy and bone transport to fill in a segmental tibial diaphyseal defect. Regenerate bone formed well in the gap that was created that trailed the transport segment, and slow healing at the docking site was observed, as seen in humans. The angiographic techniques clearly revealed, by radiography and anatomic dissection, the arterial response to bone transport. The results showed that the transport segment had an arterial supply after osteotomy and after transport. They also demonstrated an extensive increase in vessels in limbs that had undergone distraction osteogenesis, an observation made clinically in humans but not well demonstrated experimentally. Furthermore, angiography showed proximal stretching and distal kinking of the major artery of the leg. This model closely resembles distraction osteogenesis through bone transport in humans and definitively demonstrates that the transport segment can maintain blood supply and remain viable during the transport process. The results of this study provide a basis for further work on factors that enhance and interfere with successful bone transport in humans.

Leg lengthening over an intramedullary nail.

Distraction osteogenesis is widely used for leg lengthening, but often requires a long period of external fixation which carries risks of pin-track sepsis, malalignment, stiffness of the joint and late fracture of the regenerate. We present the results of 20 cases in which, in an attempt to reduce the rate of complications, a combination of external fixation and intramedullary nailing was used. The mean gain in length was 4.7 cm (2 to 8.6). The mean time of external fixation was 20 days per centimetre gain in length. All distracted segments healed spontaneously without refracture or malalignment. There were three cases of deep infection, two of which occurred in patients who had had previous open fractures of the bone which was being lengthened. All resolved with appropriate treatment. This method allows early rehabilitation, with a rapid return of knee movement. There is a lower rate of complications than occurs when external fixation is used on its own. The time of external fixation is shorter than in other methods of leg lengthening. The high risk of infection calls for caution.

The morphological basis of increased stiffness of rabbit tibialis anterior muscles during surgical limb-lengthening.

When the tibialis anterior muscle of the rabbit is progressively stretched during surgical limb distraction, the muscle fibres lengthen by addition of new serial sarcomeres, provided that stretch is carried out at an appropriate rate. However, in spite of the apparent adaptation to the new functional length, range of joint movement is greatly decreased. In this study we have first, made measurements of the passive tension developed by distracted muscles over the range of joint movement and secondly made quantitative measurements of endomysial and perimysial connective tissue content. It was found that at all ankle joint angles greater than 90 degrees, the passive tension developed by the distracted muscles was greater than both contralateral and sham-operated controls. Image analysis showed that the ratio of collagen to contractile material was increased in distracted muscles compared with muscles from sham-operated controls, due to increased deposition of collagen type III. Scanning electron microscopy showed the presence of a dense perimysial weave surrounding the distracted muscle fibres. These quantitative and qualitative changes in the connective tissue component could account for the increased stiffness demonstrated by the physiological measurements. It would seem that in distracted muscle the connective tissue element adapts less readily than the contractile component, with prolonged stretch leading to damage to the perimysial and endomysial network, with subsequent fibrosis and loss of muscle compliance. Such changes could help explain the loss of range of movement noted in the distracted limbs of patients undergoing surgical limb-lengthening and in other conditions that result in muscle contractures.

Measurement of impact force, simulation of fall and hip fracture.

It has been shown that the incidence of hip fracture in the elderly may be influenced by the type of floor covering commonly used in homes for the elderly. This study describes the development of a method for modelling a fall during a hip fracture event, to examine the influence of different floors on impact force. An impact transducer is dropped in free fall through a smooth plastic tube. The impactor nose of the transducer models the curvature of the greater trochanter, and a steel spring is used to simulate the compliance of the skeletal structure. A weight, which corresponds to one-sixteenth of average body mass, compresses the spring and applies force to the impactor nose on striking the floor. The temporal variation in the force of impact with the floor is measured by the transducer to within 0.41 percent (SD = 0.63%, n = 10). Five common floor coverings were tested over a concrete floor slab (vinyl, loop carpet and pile carpet--both with and without underpad). ANOVA analysis showed that the differences between mean forces for each floor covering were highly significant (p > 0.001), with the thicker coverings producing 7 percent lower forces. The transducer may be used to examine the correlation between impact force and fracture incidence for a variety of different floors in homes for the elderly.

Strain rate and timing of stimulation in mechanical modulation of fracture healing.

Fracture of the long bones results in a repair process that has the potential to restore the anatomic morphology and mechanical integrity of the bone without scar tissue. The repair process can occur in two patterns. In the first, under conditions of rigid stabilization, direct osteonal remodeling of the fracture line can occur with little or no external callus, a process known as direct bone repair. The second pattern of repair involves bridging of the fragments with external callus and formation of bone in the fracture site by endochondral healing. This type of repair is known as indirect bone healing and occurs under less rigid interfragmentary stabilization. The rate of healing and the extent of callus in this type of repair can be modulated by the mechanical conditions at the fracture site. Applying cyclic interfragmentary micromotion for short periods has been shown to influence the repair process significantly, and characteristics of this stimulus influence the healing response observed. In the current study, a short term interfragmentary cyclic micromovement applied at a high strain rate induced a greater amount of periosteal callus than the same stimulus applied at a low strain rate. This high strain rate stimulus applied later in the healing period significantly inhibited the progress of healing. The beneficial effect of this particular biophysic stimulus early in the healing period may be related to the viscoelastic nature of the differentiating connective tissues in the early endochondral callus. In the early endochondral callus, high rates of movement induce a greater deformation of the fracture fragments because of the stiffening of the callus. Alternatively, the transduction pathway may involve streaming potentials as a result of the high movement rate.

Mechanical influences on tibial fracture healing.

Selected studies are summarized that measure interfragmentary fracture displacements in 6 degrees of freedom at intervals throughout healing in groups of patients with tibial diaphyseal fractures treated by external skeletal fixation. The results are compared with those obtained from experimental studies in which the ideal mechanical conditions for fracture healing were predicted. A finite element analysis model of the healing tibial fracture also was developed. Measured data were used for the analysis, and stress and strain patterns were defined for different stages of healing. Interfragmentary movement measured in the first 6 weeks after injury usually is a magnitude smaller in patients treated by external fixation than in patients treated with cast immobilization. This movement can be much smaller than that predicted to be optimal by experimental studies. A greater amplitude can be achieved, even in stable fractures, by ensuring patient activity. The interfragmentary movement is elastic during loading activity and is generally sinusoidal during steady walking. At the time of dynamization (the unlocking of the frame), a permanent set occurs at the fracture site in all planes. The cyclical movement range in each plane often decreases immediately after unlocking. The model analysis study of fracture healing predicts that tissue damage may occur in the later (hard callus) phase of healing, even while the fixation device is in place, because of abnormally high stresses and strains. This study indicates that fracture mechanics should be controlled more rigorously to provide amplitudes of movement in the first 4 to 6 weeks after fracture. The rigidity of fixation should be increased in the subsequent weeks until the fracture has healed and the frame is removed.

Force-displacement behaviour of biological tissue during distraction osteogenesis.

Limb lengthening and bone transport treatments are used frequently, and complications during treatment are common. Knowledge of the origin of tensile forces which resist distraction and the force-displacement response of biological tissues during extension would assist in reducing complication rates. Two tibial diaphyseal lengthenings carried out using an Ilizarov fixator were monitored by a displacement transducer to determine extension of the lengthening tissue (the regenerate bone), and by force transducers to determine tensile forces in the tissues that resist extension. The position of the force vector within a cross-section of the limb at the regenerate (provided by CT) was used to determine the origin of these forces. The muscle groups and adjacent fascia resisting extension were the gastrocnemius in one subject and the anterior and peroneal compartments in the other. In response to distraction, these tissues had relatively high stiffness (> 200 N/mm), less "immediate" displacement (< 35% of long term non-recoverable displacement) and inconsistent force relaxation properties (0-90%). In contrast, when the force vector was located in the vicinity of the regenerate, tissue exhibited lower stiffness (< 50 N/mm) and more immediate displacement (> 65% of long term nonrecoverable displacement), but also exhibited inconsistent force relaxation (0-67%).

Prevention of deformity during limb lengthening.

Deformity occurs frequently at the site of distraction during leg lengthening and can add to disability. The elastic and nonelastic displacements have been measured in a model that simulates leg lengthening in the laboratory. Measurements have been made for different fixator systems. The angulation in the vertical plane that occurs during leg lengthening is minimized if the distance between the bone and the fixator bar is kept as small as possible, if three screws are inserted in the proximal and distal bone fragments, and if the peak loads on the fixator are reduced by adjusting the rhythm of distraction. However, even if these precautions are taken, the results show that some fixators designed for leg lengthening will fail and lead to deformity at the osteotomy site. This may occur under the repeated cycles of high loads associated with the rises in soft tissue tension that are known to occur in certain groups of patients. This study suggests that deformity can be prevented by the proper selection of a suitable frame and the adjustment of its configuration to meet the loading requirements.

A method of examining the magnitude and origin of "soft" and "hard" tissue forces resisting limb lengthening.

Complications arising from limb-lengthening procedures such as muscle contracture, axial malalignment of the bone and traction injuries to the nerves and vessels, are often severe. Often complications arise from the build-up of forces in the biological tissues which are resisting lengthening. Little is known about the origin and magnitude of these forces, although three studies have identified the regenerate (new bone tissue) as the dominant resisting tissue. This study describes the development of a method to examine these forces. It employs load measurement devices in the structural columns of Ilizarov fixators which measure the compressive load on the frame exerted by the biological tissues. The distribution of this load between the columns of the frame, in conjunction with a transverse radiograph of the limb at the regenerate site, is used to examine the origin of the resisting force. Accuracy was determined by a laboratory simulation which found the predicted position of the force to be within 5 mm of the actual position in all four cases tested. Mean error in the total measured force was 2 N (SD, 1 N). A pilot study on a patient undergoing a 60 mm femoral lengthening revealed a peak force of 717 N originating in the Vastus Lateralis or the illiotibial tract. Negligible contribution to resistance was provided by the regenerate, contrary to that found with other studies.

Temporal changes in dynamic inter fragmentary motion and callus formation in fractures.

This study examined whether callus proliferation at long bone fractures is triggered by cyclical inter-fragmentary displacement which arises from routine activity. It also examined whether a growing callus increases the stability of a fracture, thereby reducing displacement amplitude during relative motion. Seven tibial fractures stabilised with external fixators were monitored up to and beyond fixator removal. An instrumented spatial linkage was developed which was attached to the bone screws to measure inter-fragmentary displacement at the fracture in all six degrees of freedom during routine walking. Callus index (final bone width/initial bone width) was measured at the posterior and lateral cortical surfaces from orthogonal radiographs. In all seven subjects, callus growth was initiated subsequent to a peak in displacement which occurred within the first 42 days; at nine of the 14 surfaces occurred callus initiation occurred within 14 days of the peak displacement. With the exception of two lateral surfaces, maximum callus size, subsequent to fixator removal (at up to 119 days after removal). Displacement reduced during callus growth in five out of six subjects. Since the reduction in displacement did not arise from reduced weight-bearing, increasing callus size must correlate with progressive mechanical union. This was confirmed by end point stiffness tests. Therefore, peak cyclical displacement appears to be the stimulus for callus growth, the effect of which is to reduce displacement and strain which allows the following stages of bone formation and remodelling to unite the fracture.

A biomechanical study on five unilateral external fracture fixation devices.

OBJECTIVE: This study examines plastic, slip and fatigue failures in external fixator frames. DESIGN: Failure was examined in five commonly used unilateral external fixators using experimental models that simulated both stable and unstable diaphyseal fractures. BACKGROUND: Plastic failure arises from permanent deformation of a frame initiated by bending of frame components, while slip failure arises from slip at clamping interfaces. Such failure may lead to refracture or to malalignment of the bone (transverse or angular). Fatigue failure arises from the loosening or wear of components under long-term cyclic loading; this can lead to variable interfragmentary displacement and impairment of fracture stability. METHODS: Axial loads were measured at which plastic or slip failure occurred, as were changes in interfragmentary motion due to 10 000 load cycles simulating walking activity. RESULTS: In four fixators, plastic or slip failure initiated bone malalignment at only 50% of average adult weight bearing (650 N). Additionally, in three fixators a third screw at the clamp centre was found to reduce retention of the two adjacent screws, causing premature slip failure. Evidence of fatigue was found in all the fixators after only 10 000 load cycles. In one fixator, the amplitude of elastic interfragmentary motion increased progressively by 30% transversely, 15% angularly and 100% torsionally. In another fixator it increased by 10% in all directions while the column was locked (non-dynamizing), and it increased a further 10% while it was unlocked and able to slide telescopically (dynamizing). Clamp slip occurred at the column of a third fixator almost immediately after commencing the cyclic loading. CONCLUSION: Plastic or slip failure of frames may occur prematurely during routine weight-bearing on unstable fractures, and frame fatigue may affect long-term interfragmentary stability.