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.

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 the new bone zone 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 the 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.

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.

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.

Scoliosis associated with limb-length inequality.

We reviewed the records and physical findings of twenty-three young adults who had had significant untreated limb-length inequality, present since childhood. The spine was studied radiographically and clinically before and after neutralization of the discrepancy of limb lengths with a lift. Movements of the spine also were measured. A significant asymmetry of lateral flexion of the spine remained in nearly all of the patients after neutralization of the discrepancy. The lumbar scoliosis associated with the limb-length inequality was compensatory and non-progressive, but abnormalities of the Cobb angle and of axial rotation remained in the young adults. No relationship was found between the underlying cause of the anisomelia or its duration and the severity of the spinal abnormality. The scoliosis was minor in patients with discrepancies of less than 2.2 centimeters. No patients complained of significant discomfort in the back, nor were degenerative radiographic changes evident there.

The role of fixator frame stiffness in the control of fracture healing. An experimental study.

External skeletal fixation is used widely in the management of fractures. Frame configuration is known to affect frame stiffness and, thereby, the local mechanical environment at the fracture site. In previous investigations of the influence of mechanical conditions upon fracture healing, the frames have always been applied so that they influence the biological environment in different ways. As a result, the influence of stiffness, per se, could not be studied as a single variable, and its effect on the repair process remains unclear. In this study, using a standard osteotomy of the ovine tibia, stabilised by an external skeletal fixator, the local mechanical environment was altered solely by increasing the 'offset' distance between the bone and the fixator frame. The biological conditions at the fracture remained identical in both groups. Increasing the frame stiffness by 40%, brought about by reducing the offset distance of the fixator bar by 10 mm, caused a significant reduction in the rate of healing. In addition, the frame stiffness influenced the ground reaction force with greater weight-bearing in the initial stages in the more rigid group, but despite this, the resultant interfragmentary displacement in this group appeared to be insufficient to stimulate fracture healing. This work emphasises the importance of the local mechanical environment on the process of fracture healing. It also demonstrates the value of in vivo assessment of fracture stiffness as a means of monitoring mechanical events during fracture healing.

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.

Problems encountered in leg shortening.

We reviewed 46 leg-shortening operations (37 femoral and nine tibial), performed by different methods, to assess the incidence of complications and permanent disability. Shortening of as much as 7.5 cm in the femur and 5 cm in the tibia was achieved in men of normal height without any loss of function. Complications were seen with all surgical techniques despite the use of modern implants. Most problems arose from inadequate stabilisation of the osteotomy. The most reliable method of femoral shortening was open subtrochanteric osteotomy with preservation of the isthmus, and fixation with an intramedullary nail locked at its proximal end. In tibial shortening, bone excision should be at the level of the flare in the lower diaphysis in order to achieve reliable bone healing. Simple intramedullary nail fixation should be supplemented with a long-leg cast for six weeks or the nail should be locked at both ends to prevent postoperative distraction or rotation.

Physeal distraction and cell proliferation in the growth plate.

We studied the cellular response to physeal distraction in the growth plates of skeletally immature rabbits. We used a new method of labelling and detection of proliferating cells with bromodeoxyuridine (BUdR) and an anti-BUdR antibody. The application of an external fixator but no distraction force produced no changes in the growth plates. After five days of distraction at a maximum force of 20 N, the growth plate became thicker, mainly because of an increase in the number of hypertrophic chondrocytes, but there was no evidence of increased cell proliferation. Recent fractures were seen at the junction of growth plate and metaphysis but the increase in bone length was insignificant. After ten days of distraction at the same maximum force, the chondrocyte columns had become disorganised and cell proliferation was significantly decreased. There was an increase in bone length due to distraction of the fracture gap. In this model, physeal distraction did not stimulate cell proliferation, but actually inhibited it. The apparent increase in growth-plate thickness produced by distraction is not due to increased cell production, but results from inhibition of endochondral ossification and the consequent accumulation of hypertrophic chondrocytes. Any growth after distraction depends on the ability of growth-plate chondrocytes to divide. The decrease in proliferative activity which we found after ten days of distraction suggests the need for caution in the use of such procedures in young children.

The influence of induced micromovement upon the healing of experimental tibial fractures.

Although it has been well established that fracture healing is influenced by the mechanical environment, the optimal parameters have not yet been established. In two groups of sheep an experimental tibial diaphysial fracture was created, and stabilised using external skeletal fixation. In one group rigid fixation was maintained throughout fracture healing; in the other group controlled axial micromovement, with a loading regime known to be osteogenic in intact bones, was applied for a short period daily. A significant improvement in healing was associated with the application of controlled micromovement. Data from these experiments provide the basis for improving the conditions for fracture healing and may assist in the prevention of delayed union.

The timing of distraction of an osteotomy.

New methods of limb lengthening are being adopted in the hope of overcoming the poor osteogenic responses characteristic of distraction. Delay between the osteotomy and starting distraction is said to be important but there is little experimental evidence. We have compared immediate with delayed distraction in the rabbit tibia and shown that delay is an important factor in promoting osteogenesis. It seems that its effects are partly mediated by an improvement in the extra-osseous blood supply.

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.

One-stage lengthening for femoral shortening with associated deformity.

One-stage femoral lengthening is thought to have an unacceptably high complication rate and is not widely practised. We reviewed 17 patients after one-stage lengthening for femoral shortening with associated angular or rotational deformities. Minimal dissection of the bone ends was undertaken. The mean length gain was 4 cm (2 to 7), and the average time to union was 6 months (3 to 10). There were no neurovascular complications. Four patients had delayed or nonunion, but union was achieved after bone grafting. We conclude that with minimal dissection, and with iliac crest cancellous bone grafting, one-stage leg lengthening for correction of deformity and leg-length inequality of up to 7 cm, in selected patients, can be effected safely with a relatively short rehabilitation.

The forces which develop in the tissues during leg lengthening. A clinical study.

Axial forces were measured during limb lengthening in a series of ten patients with varying pathologies in order to assess the mechanical characteristics of the distracted tissues and the levels of axial force to which soft tissues are subjected during leg lengthening. The pattern of force was found to vary according to the underlying pathology. For post-traumatic shortening in adults both the peak and the resting forces rose steadily during lengthening reaching maximum forces of the order of 300 N. Patients with congenitally short limbs developed very high peak forces (in some cases over 1000 N) and also showed large amounts of force relaxation (typically 400 to 500 N). When very high levels of force were recorded, there was a higher complication rate. In particular, there was a high instance of angular deformity. This occurred because the loads encountered resulted in failure of some of the external fixation frames.

The influence on the spine of leg-length discrepancy after femoral fracture.

We investigated the spines of 15 patients who had significant leg-length inequality as a result of femoral shaft fractures sustained after skeletal maturity but below the age of 21 years. The patients were examined at least 10 years after fracture. The spines were studied clinically and radiographically before and after correction of leg-length inequality with a shoe-raise. Lateral spinal flexion was measured from radiographs. The lumbar scoliosis associated with the leg-length inequality was compensatory: after equalisation of leg-length the overall curve and the axial rotation were corrected completely. There was also an equal range of lateral flexion to either side after correction. Minor malalignments of the whole spine remained despite correction of the compensatory scoliosis, and within the lumbar spine correction of the scoliosis had not occurred equally at all levels. No patients complained of significant discomfort and neither structural abnormalities nor degenerative changes were seen on the radiographs.

Leg-length inequality in children treated by Syme's amputation.

We have reviewed the results of amputation through the ankle in the management of 37 children with congenital leg-length discrepancy, followed up for a mean of 7.6 years after operation. In general good function was achieved and 18 patients considered their activities to be unrestricted. The main factor affecting the functional result was the underlying condition for which operation had been performed. Although heel pad migration, scar rotation and os calcis remnants were seen, these could be accommodated by the prosthesis. Syme's amputation is tolerated well in the younger child and, in patients with a predicted leg-length discrepancy of over 15 cm associated with an abnormal foot, we recommend the operation as a primary procedure between the ages of 18 months and two years.

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.

Dynamisation of tibial fractures.

We studied the effect of 'dynamisation' on tibial fractures in six patients treated by the Dynamic Axial Fixator. In the early stages, peak cyclic movement at two to four weeks averaged 0.75 mm (0.19 to 1.02) on the medial side of the bone and 0.86 mm (0.21 to 1.25) on the lateral side. The amount of movement correlated with the applied load and the fracture stiffness. After unlocking the fixator column at six weeks, progressive closure of the gap averaged 1.3 mm (0.1 to 3.5). Cyclic movement is produced by early weight-bearing with the fixator column locked. Progressive closure occurs after unlocking the column, and is often associated with a reduction in cyclic movements. The effects of dynamisation on movement at the fracture site should be defined separately, in terms of cyclic movement and of progressive closure.

The response of muscle to leg lengthening.

We used an experimental rabbit model of leg lengthening to study the morphology and function of muscle after different distraction rates. Lengthening was in twice-daily increments from 0.4 to 4 mm per day. New contractile tissue formed during lengthening, but some damage to the muscle fibres was seen even at rates of less than 1 mm per day; abnormalities increased with larger rates of lengthening. There was proliferation of fibrous tissue between the muscle fibres at distraction rates of over 1 mm per day. Active muscle function showed adaptation when the rate was 1.0 mm per day or less, but muscle compliance was normal only after rates of 0.4 mm per day. Muscle responded more favourably at rates of distraction slower than those shown to lead to the most prolific bone formation. At present the rate of distraction in clinical practice is determined mainly by factors which enhance osteogenesis. Our study suggests that it may be advisable to use a slower rate of elongation in patients with poor muscle compliance associated with the underlying pathology; this will allow better accommodation by the contractile and connective tissues of the muscles.

Measuring stiffness can define healing of tibial fractures.

We measured fracture stiffness in 212 patients with tibial fractures treated by external fixation. In the first 117 patients (group 1) the decision to remove the fixator and allow independent weight-bearing was made on clinical grounds. In the other 95 patients (group 2) the frames were removed when the fracture stiffness had reached 15 Nm/degree. In group 1 there were eight refractures and in group 2 there was none (p = 0.02, Fisher's exact test). The time to independent weight-bearing was longer in group 1 (median 24 weeks) than in group 2 (21.7 weeks, p = 0.02). The greater precision of our objective measurement was associated with a reduction in refracture rate and in the time taken to achieve independent weight-bearing. We consider that a stiffness of 15 Nm/degree in the sagittal plane provides a useful definition of union of tibial fractures.