Marina R. Makarov

Citric acid-derived in situ crosslinkable biodegradable polymers for cell delivery

Herein, we report a first citric acid (CA)-derived in situ crosslinkable biodegradable polymer, poly(ethylene glycol) maleate citrate (PEGMC). The synthesis of PEGMC could be carried out via a one-pot polycondensation reaction without using organic solvents or catalysts. PEGMC could be in situ crosslinked into elastomeric PPEGMC hydrogels. The performance of hydrogels in terms of swelling, degradation, and mechanical properties were highly dependent on the molar ratio of monomers, crosslinker concentration, and crosslinking mechanism used in the synthesis process. Cyclic conditioning tests showed that PPEGMC hydrogels could be compressed up to 75% strain without permanent deformation and with negligible hysteresis. Water-soluble PEGMC demonstrated excellent cytocompatibilty in vitro. The degradation products of PPEGMC also showed minimal cytotoxicity in vitro. Animal studies in rats clearly demonstrated the excellent injectability of PEGMC and degradability of the in situ-formed PPEGMC. PPEGMC elicited minimal inflammation in the early stages post-injection and was completely degraded within 30 days in rats. In conclusion, the development of CA-derived injectable biodegradable PEGMC presents numerous opportunities for material innovation and offers excellent candidate materials for in situ tissue engineering and drug delivery applications.

Histomorphometry of Distraction Osteogenesis in a Caprine Tibial Lengthening Model

Standardized histomorphometry of bone formation and remodeling during distraction osteogenesis (DO) has not been well characterized. Increasing the rhythm or number of incremental lengthenings performed per day is reported to enhance bone formation during limb lengthening. In 17 skeletally immature goats, unilateral tibial lengthenings to 20 or 30% of original length were performed at a rate of 0.75 mm/day and rhythms of 1, 4, or 720 times per day using standard Ilizarov external fixation and an autodistractor system. Two additional animals underwent frame application and osteotomy without lengthening and served as osteotomy healing controls. Histomorphometric indices were measured at predetermined regions from undecalcified tibial specimens. Within the distraction region, bone formation and remodeling activity were location dependent. Intramembranous bone formed linearly oriented columns of interconnecting trabecular plates of woven and lamellar type bone. Total new bone volume and bone formation indices were significantly increased within the distraction and osteotomy callus regions (Tb.BV/TV, 226% [p < 0.05]; BFR/BS, 235–650% [p < 0.01]) respectively, compared with control metaphyseal bone. Bone formation indices were greatest adjacent to the mineralization zones at the center of the distraction gap; mineral apposition rate 96% (p < 0.01); mineralized bone surfaces 277% [p < 0.001]); osteoblast surfaces 359% [p < 0.001]); and bone formation rate (650% [p < 0.01]). There was no significant difference (p < 0.14; R = 0.4) in the bone formation rate of the distracted callus compared with the osteotomy control callus. Within the original cortices of the lengthened tibiae, bone remodeling indices were significantly increased compared with osteotomy controls; activation frequency (200% [p < 0.05]); osteoclast surfaces (295% [p < 0.01]); erosion period (75%); porosity (240% [p < 0.001]). Neither the rhythm of distraction nor the percent lengthening appeared to significantly influence any morphometric parameter evaluated. Distraction osteogenesis shares many features of normal fracture gap healing. The enhanced bone formation and remodeling appeared to result more from increased recruitment and activation of bone forming and resorbing cells rather than from an increased level of individual cellular activity.

Evaluation of inferior alveolar nerve function during distraction osteogenesis in the dog

PURPOSE A series of electrophysiologic studies were performed in a canine model to evaluate inferior alveolar nerve (IAN) function during distraction osteogenesis of the mandible. MATERIALS AND METHODS Fourteen dogs, including two controls, were used in the study. Twelve dogs underwent a 10-mm bilateral mandibular lengthening with an intraoral bone-borne appliance and midbody osteotomy. By using sensory nerve action potentials, IAN function was assessed before and immediately after surgery, before and at the completion of distraction, and before necropsy after 4, 6, or 8 weeks of fixation. RESULTS Twelve of the 24 nerves showed a complete loss of evoked potential after surgery without recovery at any point throughout the study. Acute nerve injury caused by either the osteotomy or screw encroachment was identified at necropsy. The other 12 nerves showed reproducible responses after surgery. Eight of these nerves had significant amplitude attenuation of the evoked potentials, which was identified at necropsy as a result of acute injury. The remaining four nerves did not show significant evoked potential abnormalities and appeared to be grossly normal at necropsy. During distraction, the amplitude of evoked potentials in all 12 nerves remained at the postoperative level, whereas latency showed a significant delay. In 7 of these 12 nerves, various degrees of evoked potential recovery were identified at the completion of the study. CONCLUSIONS The high incidence of acute IAN injury in the current study was primarily related to device construction and osteotomy technique. If acute nerve injury is avoided at surgery, distraction osteogenesis with 10 mm mandibular lengthening appears to produce minimal deleterious effect on IAN function.

The effect of the amount of limb lengthening on skeletal muscle.

The adaptation of tibialis anterior muscles after 20% and 30% gradual limb lengthening was evaluated. Eight skeletally mature neutered male goats had 20% (n = 4) or 30% (n = 4) tibial distraction at a rate of 0.25 mm three times per day. Muscles from lengthened and contralateral control limbs were harvested on completion of distraction. Fiber length and sarcomere length were measured followed by calculation of sarcomere number and muscle fiber-to-bone lengthening ratio. Fiber length and sarcomere number after 20% and 30% limb lengthening were significantly greater in the distracted muscles, whereas no difference in sarcomere length was detected. The difference in muscle fiber length and sarcomere number between distracted and control limbs was greater in the 30% than in the 20% group. The disproportion between the amounts of muscle fiber and bone length increase was similar after 20% and 30% lengthening. The results show that muscular adaptation continues during 20% to 30% limb lengthening by increasing fiber length. It seems that this increase occurs through serial sarcomere addition rather than sarcomere length alteration. The higher rate of musclerelated clinical complications after limb lengthening beyond 20% does not seem to be related to a failure of muscle fiber contractile elements to adapt to increasing limb length.

Effect of rhythm and level of distraction on muscle structure: an animal study.

To determine the optimal rhythm of distraction and level of osteotomy for maximal muscle preservation during limb lengthening, histologic and ultrastructural analysis of the peroneus tertius muscle was performed in 46 goats. Thirty-nine animals with 20% or 30% tibial lengthening at the rate of 0.75 mm per day at each osteotomy level were divided in two groups. The first group included 15 animals undergoing tibial middiaphyseal lengthening with automated distractor producing one (n = 5), four (n = 5), or 720 (n = 5) increments per day. The second group included 24 animals in which lengthening was performed at the proximal (n = 4), middle (n = 6), distal (n = 6), or double (n = 8) level of the tibia with distraction rhythm of three times per day. The remaining seven animals served as controls. Histomorphologic features of the lengthened muscles were characterized by a polymorphous appearance with foci of necrosis, atrophy, and sclerosis scattered among apparently normal muscle fibers. The intensity and dispersion of degenerative changes were in direct proportion to the amount of lengthening but in reverse proportion to the frequency of distraction. Tibial lengthening at the diaphysis caused intense fibroblast proliferation and remarkable muscle sclerosis, whereas metaphyseal distraction was associated with a predominance of muscle tissue regeneration over atrophy and sclerosis. Bifocal lengthening and a more fractionated rhythm of distraction were associated with enhanced preservation of existing muscle fibers and greater regenerative activity of the muscle.

Effects of external fixation and limb lengthening on peripheral nerve function

To identify factors affecting peripheral nerve function during limb lengthening, serial somatosensory evoked potentials studies were performed in 18 goats and correlated with gross appearance of the soft tissues at necropsy. In 15 goats, a 20% or 30% tibial lengthening was done using the Ilizarov apparatus at a rate of 0.75 mm per day and rhythm of 1, 4, or 720 times per day. Three animals served as frame/corticotomy controls. Seven lengthened and 2 control animals maintained normal somatosensory evoked potentials throughout the study. At necropsy, the peroneal and tibial nerves appeared grossly normal. In one control animal with irreversible loss of evoked potentials documented intraoperatively, peroneal nerve impalement by a transfixion wire was identified at necropsy. Eight lengthened animals experienced significant changes of peroneal nerve conduction. In 6 of these 8 animals, transfixion wires were found to be disrupting the nerve in some way. In the remaining 2 animals, no offending wires were identified, but there was extensive diffuse soft tissue fibrosis within the extremity. The rhythm of distraction did not have an important influence on evoked potential changes during the course of distraction. Although there was a correlation between the amount of lengthening performed and the degree of evoked potentials deterioration, the anatomic relationship between the wires and nerves was a more important factor in the development of these abnormalities.

Monitoring peripheral nerve function during external fixation of upper extremities

Limb lengthening of the upper extremity using external fixation devices is associated with a high risk of neurovascular impairment. To aid early detection of nerve injury, intraoperative monitoring of neural function was performed in five patients undergoing Ilizarov-type circular external-fixator application. The apparatus was applied to the humerus in two cases and to the forearm in the other three cases. The function of ulnar, median, and radial nerves was assessed continuously throughout the surgery by using sensory-evoked potential (SEP) monitoring technique. The responses were elicited by stimulation of these nerves in the distal forearm with recording at Erbs point and over the anterior cervical spine. During apparatus application, radial-response attenuation was identified in two instances. No corrective actions were performed in one case, and the patient had symptoms of radial nerve deficit postoperatively. In the other case, prompt removal of the offending half-pin after the detection of electrophysiologic abnormalities resulted in subsequent response restoration and the avoidance of postoperative nerve dysfunction. Sensory-evoked potential monitoring proved to be a reliable indicator of nerve compromise during external fixation of upper extremities and may be considered a valuable surgical adjunct.

Changes in pennate muscle architecture after gradual tibial lengthening in goats

The purpose of this investigation was to examine the changes in unipennate muscle architecture after distraction osteogenesis. Nine adult goats underwent 20% tibial lengthening in one of the hind limbs. Immediately after distraction, lengthened and contralateral (untreated) tibialis caudalis (TC) muscles were harvested. Lengths of the muscle belly, muscle fiber (FL), sarcomere (SL), tendon (TL), and superficial aponeurosis, as well as muscle mass, pennation angle (PA), and physiological cross‐sectional area (PCSA), were compared between the treated and contralateral sides. Lengthened TC muscle demonstrated 20.8% increase in belly length, 4.39% increase in TL, and 36.7% increase in FL, while PA decreased by 37.2% (P = 008). Muscle length increase was mainly due to lengthening of muscle belly, which resulted both from FL increase and 15.3% length increase in the aponeurosis component of muscle belly, without significant effect of the PA decrease. The FL increase was due to SL increase, not to sarcomere neogenesis, while mass and PCSA did not change. We concluded that although muscle architecture can be adversely affected by distraction because of deficient sarcomere neogenesis, PCSA can remain unchanged, giving false impression of preserved function. Change in PA plays only minimal role in muscle adaptation to distraction. Anat Rec, 2007.

Somatosensory evoked potential monitoring of peripheral nerves during external fixation for limb lengthening and correction of deformity in children

We undertook a retrospective analysis of 306 procedures on 233 patients, with a mean age of 12 years (1 to 21), in order to evaluate the use of somatosensory evoked potential (SSEP) monitoring for the early detection of nerve compromise during external fixation procedures for limb lengthening and correction of deformity. Significant SSEP changes were identified during 58 procedures (19%). In 32 instances (10.5%) the changes were transient, and resolved once the surgical cause had been removed. The remaining 26 (8.5%) were analysed in two groups, depending on whether or not corrective action had been performed in response to critical changes in the SSEP recordings. In 16 cases in which no corrective action was taken, 13 (81.2%, 4.2% overall) developed a post-operative neurological deficit, six of which were permanent and seven temporary, persisting for five to 18 months. In the ten procedures in which corrective action was taken, four patients (40%, 1.3% overall) had a temporary (one to eight months) post-operative neuropathy and six had no deficit. After appropriate intervention in response to SSEP changes, the incidence and severity of neurological deficits were significantly reduced, with no cases of permanent neuropathy. SSEP monitoring showed 100% sensitivity and 91% specificity for the detection of nerve injury during external fixation. It is an excellent diagnostic technique for identifying nerve lesions when they are still highly reversible.

Acute deformity correction of lower extremities under SSEP-monitoring control.

Intraoperative somatosensory evoked potential (SSEP) monitoring was performed in eight children who had undergone an acute deformity correction in the lower extremities using external fixation. Five patients showed stable evoked potentials during surgery and had no neurologic complications postoperatively. Three patients experienced evoked potential abnormalities. In one patient, 60° external rotation of the foot produced significant SSEP changes. The reduction of rotation to 40° resulted in tibial but not peroneal SSEP recovery. Peroneal nerve deficit was noted postoperatively. The second patient showed substantial SSEP attenuation after 45° correction of distal tibial valgus. However, spontaneous recovery of the response occurred, which allowed maintenance of the achieved correction. In a third patient, significant SSEP changes occurred after 90° external rotation and 10 mm medial translation of the distal femur. Total release of translation allowed 75° external rotation without SSEP abnormalities. Neither of the latter two patients had peripheral nerve deficits postoperatively. Intraoperative SSEP monitoring thus helps to define a neurologically safe limit of acute deformity correction.