ALM Therapy Promotes Functional and Histologic Regeneration of Traumatized Peripheral Skeletal Muscle.

Skeletal muscle trauma is a common injury with a range of severity. Adenosine, lidocaine and Mg2+ (ALM) is a protective solution and improves tissue perfusion and coagulopathy. Male Wistar rats were anesthetized and subjected to standardized skeletal muscle trauma of the left soleus muscle with the protection of the neurovascular structures. Seventy animals were randomly assigned to saline control or ALM. Immediately after trauma, a bolus of ALM solution was applied intravenously, followed by a one-hour infusion. After 1, 4, 7, 14 and 42 days, the biomechanical regenerative capacity was examined using incomplete tetanic force and tetany, and immunohistochemistry was used to examine for proliferation and apoptosis characteristics. Biomechanical force development showed a significant increase following ALM therapy for incomplete tetanic force and tetany on days 4 and 7. In addition, the histological evaluation showed a significant increase in proliferative BrdU-positive cells with ALM therapy on days 1 and 14. Ki67 histology also detected significantly more proliferative cells on days 1, 4, 7, 14 and 42 in ALM-treated animals. Furthermore, a simultaneous decrease in the number of apoptotic cells was observed using the TUNEL method. ALM solution showed significant superiority in biomechanical force development and also a significant positive effect on cell proliferation in traumatized skeletal muscle tissue and reduced apoptosis.

[Functional rehabilitation after transfemoral amputation : Shaft prosthesis or endo-exo prosthesis?] / Funktionelle Rehabilitation nach Oberschenkelamputation : Schaftprothese oder Endo-Exo-Prothese?

BACKGROUND: After transfemoral amputation a prosthesis is required to restore autonomous standing and bipedal locomotion. Attachment of the prosthesis can be achieved either classically via socket suspension with a shaft in the stump or directly via implantation of an intramedullary transcutaneous femoral prosthesis (osseointegrated prosthesis). AIM: A fully instrumented gait analysis should enable objectification of the anticipated advantages of the EEP with respect to the gait pattern and individual mobility. MATERIAL AND METHODS: In two patients with a unilateral transfemoral amputation a comprehensive gait analysis was carried out prior to and 6 months (patient 1) or 11 and 20 months (patient 2) after switching from a socket prosthesis to an EEP. This was carried out in the Gait Realtime Analysis Interactive Lab (GRAIL), a fully instrumented gait laboratory with virtual reality and enables assessment close to the conditions of daily life. RESULTS: In both cases the gait analysis confirmed the advantages associated with an EEP for the transmission of force to the prosthesis and the accompanying improvement in gait symmetry.

Cementless Titanium Mesh Fixation of Osteoporotic Burst Fractures of the Lumbar Spine Leads to Bony Healing: Results of an Experimental Sheep Model.

INTRODUCTION: Current treatment strategies for osteoporotic vertebral compression fractures (VCFs) focus on cement-associated solutions. Complications associated with cement application are leakage, embolism, adjacent fractures, and compromise in bony healing. This study comprises a validated VCF model in osteoporotic sheep in order to (1) evaluate a new cementless fracture fixation technique using titanium mesh implants (TMIs) and (2) demonstrate the healing capabilities in osteoporotic VCFs. METHODS: Twelve 5-year-old Merino sheep received ovariectomy, corticosteroid injections, and a calcium/phosphorus/vitamin D-deficient diet for osteoporosis induction. Standardized VCFs (type AO A3.1) were created, reduced, and fixed using intravertebral TMIs. Randomly additional autologous spongiosa grafting (G1) or no augmentation was performed (G2, n = 6 each). Two months postoperatively, macroscopic, micro-CT and biomechanical evaluation assessed bony consolidation. RESULTS: Fracture reduction succeeded in all cases without intraoperative complications. Bony consolidation was proven for all cases with increased amounts of callus development for G2 (58.3%). Micro-CT revealed cage integration. Neither group showed improved results with biomechanical testing. CONCLUSIONS: Fracture reduction/fixation using TMIs without cement in osteoporotic sheep lumbar VCF resulted in bony fracture healing. Intravertebral application of autologous spongiosa showed no beneficial effects. The technique is now available for clinical use; thus, it offers an opportunity to abandon cement-associated complications.

Minimum cement volume required in vertebral body augmentation--A biomechanical study comparing the permanent SpineJack device and balloon kyphoplasty in traumatic fracture.

BACKGROUND: Minimally invasive treatment of vertebral fractures is basically characterized by cement augmentation. Using the combination of a permanent implant plus cement, it is now conceivable that the amount of cement can be reduced and so this augmentation could be an attractive opportunity for use in traumatic fractures in young and middle-aged patients. The objective of this study was to determine the smallest volume of cement necessary to stabilize fractured vertebrae comparing the SpineJack system to the gold standard, balloon kyphoplasty. METHODS: 36 fresh frozen human cadaveric vertebral bodies (T11-L3) were utilized. After creating typical compression wedge fractures (AO A1.2.1), the vertebral bodies were reduced by SpineJack (n=18) or kyphoplasty (n=18) under preload (100N). Subsequently, different amounts of bone cement (10%, 16% or 30% of the vertebral body volume) were inserted. Finally, static and dynamic biomechanical tests were performed. FINDINGS: Following augmentation and fatigue tests, vertebrae treated with SpineJack did not show any significant loss of intraoperative height gain, in contrast to kyphoplasty. In the 10% and 16%-group the height restoration expressed as a percentage of the initial height was significantly increased with the SpineJack (>300%). Intraoperative SpineJack could preserve the maximum height gain (mean 1% height loss) better than kyphoplasty (mean 16% height loss). INTERPRETATION: In traumatic wedge fractures it is possible to reduce the amount of cement to 10% of the vertebral body volume when SpineJack is used without compromising the reposition height after reduction, in contrast to kyphoplasty that needs a 30% cement volume.

Acromioclavicular joint dislocations: radiological correlation between Rockwood classification system and injury patterns in human cadaver species.

INTRODUCTION: The classification system of Rockwood and Young is a commonly used classification for acromioclavicular joint separations subdividing types I-VI. This classification hypothesizes specific lesions to anatomical structures (acromioclavicular and coracoclavicular ligaments, capsule, attached muscles) leading to the injury. In recent literature, our understanding for anatomical correlates leading to the radiological-based Rockwood classification is questioned. The goal of this experimental-based investigation was to approve the correlation between the anatomical injury pattern and the Rockwood classification. MATERIALS AND METHODS: In four human cadavers (seven shoulders), the acromioclavicular and coracoclavicular ligaments were transected stepwise. Radiological correlates were recorded (Zanca view) with 15-kg longitudinal tension applied at the wrist. The resulting acromio- and coracoclavicular distances were measured. RESULTS: Radiographs after acromioclavicular ligament transection showed joint space enlargement (8.6 ± 0.3 vs. 3.1 ± 0.5 mm, p < 0.05) and no significant change in coracoclavicular distance (10.4 ± 0.9 vs. 10.0 ± 0.8 mm). According to the Rockwood classification only type I and II lesions occurred. After additional coracoclavicular ligament cut, the acromioclavicular joint space width increased to 16.7 ± 2.7 vs. 8.6 ± 0.3 mm, p < 0.05. The mean coracoclavicular distance increased to 20.6 ± 2.1 mm resulting in type III-V lesions concerning the Rockwood classification. CONCLUSIONS: Trauma with intact coracoclavicular ligaments did not result in acromioclavicular joint lesions higher than Rockwood type I and II. The clinical consequence for reconstruction of low-grade injuries might be a solely surgical approach for the acromioclavicular ligaments or conservative treatment. High-grade injuries were always based on additional structural damage to the coracoclavicular ligaments. Rockwood type V lesions occurred while muscle attachments were intact.

Radiographic and safety details of vertebral body stenting: results from a multicenter chart review.

BACKGROUND: Up to one third of BKP treated cases shows no appreciable height restoration due to loss of both restored height and kyphotic realignment after balloon deflation. This shortcoming has called for an improved method that maintains the height and realignment reached by the fully inflated balloon until stabilization of the vertebral body by PMMA-based cementation. Restoration of the physiological vertebral body height for pain relief and for preventing further fractures of adjacent and distant vertebral bodies must be the main aim for such a method. A new vertebral body stenting system (VBS) stabilizes the vertebral body after balloon deflation until cementation. The radiographic and safety results of the first 100 cases where VBS was applied are presented. METHODS: During the planning phase of an ongoing international multicenter RCT, radiographic, procedural and followup details were retrospectively transcribed from charts and xrays for developing and testing the case report forms. Radiographs were centrally assessed at the institution of the first/senior author. RESULTS: 100 patients (62 with osteoporosis) with a total of 103 fractured vertebral bodies were treated with the VBS system. 49 were females with a mean age of 73.2 years; males were 66.7 years old. The mean preoperative anterior-middle-posterior heights were 20.3-17.6-28.0 mm, respectively. The mean local kyphotic angle was 13.1°. The mean preoperative Beck Index (anterior edge height/posterior edge height) was 0.73, the mean alternative Beck Index (middle height/posterior edge height) was 0.63. The mean postoperative heights were restored to 24.5-24.6-30.4 mm, respectively. The mean local kyphotic angle was reduced to 8.9°. The mean postoperative Beck Index was 0.81, the mean alternative one was 0.82. The overall extrusion rate was 29.1%, the symptomatic one was 1%. In the osteoporosis subgroup there were 23.8% extrusions. Within the three months followup interval there were 9% of adjacent and 4% of remote new fractures, all in the osteoporotic group. CONCLUSIONS: VBS showed its strengths especially in realignment of crush and biconcave fractures. Given that fracture mobility is present, the realignment potential is sound and increases with the severity of preoperative vertebral body deformation.

Antithrombin reduces inflammation and microcirculatory perfusion failure in closed soft-tissue injury and endotoxemia.

BACKGROUND: Closed soft-tissue trauma leads to activation of the coagulation cascade and is often complicated by systemic inflammation and infection. Previous investigations have shown potent anti-inflammatory properties of antithrombin. We herein report on the action of antithrombin on skeletal muscle injury in experimental endotoxemia. MATERIALS AND METHODS: By using a pneumatically driven computer-controlled impact device, closed soft-tissue trauma was applied on the left hind limb of pentobarbital-anesthetized rats. Six hours later, endotoxemia was induced by intraperitoneal injection of Escherichia coli lipopolysaccharide. An equivalent volume of physiological saline was given in controls. At the same time point, treatment of animals was started by intravenous injection of antithrombin (250 IU/kg body weight) or vehicle solution. Twenty-four hours after trauma, the extensor digitorum longus muscle was microsurgically exposed and analyzed by means of high-resolution multifluorescence microscopy. RESULTS: Traumatic soft-tissue injury with additional endotoxemia was characterized by nutritive perfusion failure (functional capillary density: 379±20cm/cm;), tissue hypoxia (nicotinamide adenine dinucleotide autofluorescence: 77±4 aU), and enhanced leukocyte-endothelial cell interaction (773±35 cells/mm;). Therapeutic intervention with antithrombin 6 hrs after trauma restored nutritive perfusion and tissue oxygenation (functional capillary density: 469±22cm/cm; nicotinamide adenine dinucleotide autofluorescence: 61±5 aU [p < 0.05]) and reduced inflammatory leukocyte adherence (237±20 cells/mm; [p < 0.05]) toward values found in nontraumatized controls (functional capillary density: 573±13cm/cm; nicotinamide adenine dinucleotide autofluorescence: 56±2 aU; leukocyte adherence: 204±20 cells/mm;). CONCLUSION: Antithrombin ameliorates microcirculatory dysfunction and tissue injury in traumatized animals during endotoxemia. Furthermore, a reduced inflammatory cell response helps to prevent leukocyte-dependent secondary tissue injury.

Vitamin D increases cellular turnover and functionally restores the skeletal muscle after crush injury in rats.

Insufficient skeletal muscle regeneration after injury often impedes the healing process and is accompanied by functional deficiencies or pain. The aim of our study was to provide evidence that vitamin D improves muscle healing after muscle injury. Therefore, we used male rats and induced an injury of the soleus muscle. After crush injury, animals received either 8.3 mg/kg (332,000 IU/kg) body weight vitamin D or vehicle solution, s.c. After assessment of muscle force at days 1, 4, 14, and 42 after injury, sampling of muscle tissue served for analysis of proliferation, apoptosis, satellite cells, and prolyl-4-hydroxylase-ß expression. Vitamin D application caused a significant increase in cell proliferation and a significant inhibition of apoptosis at day 4 after injury compared to control animals. The numbers of satellite cells were not influenced by the vitamin D application, but there was an increase in prolyl-4-hydroxylase-ß expression, indicative of increased extracellular matrix proteins. This cellular turnover resulted in a faster recovery of contraction forces at day 42 in the vitamin D group. Current data support the hypothesis that vitamin D promotes the regenerative process in injured muscle. Thus, vitamin D treatment may represent a promising therapy to optimize recovery after injury.

Impact of diabetes and peripheral arterial occlusive disease on the functional microcirculation at the plantar foot.

BACKGROUND: Plastic and reconstructive surgeons are commonly faced with chronic ulcerations and consecutive wound infections of the feet as complications in patients with diabetes and/or peripheral arterial occlusive disease (PAOD). Microcirculatory changes seem to play an important role. However, the evaluation of functional changes in the soft tissue microcirculation at the plantar foot using combined Laser-Doppler and Photospectrometry System has not yet been performed in patients with DM or PAOD. METHODS: A prospective, controlled cohort study was designed consisting of a total of 107 subjects allocated to 1 of 3 groups-group A: healthy subjects (57% males, 63.3 y); group B: patients with diabetes mellitus (DM) (53% males, 59.4 y); and group C: patients with PAOD (81% males, 66.1 y). Microcirculatory data were assessed using a combined Laser-Doppler and Photospectrometry System. RESULTS: Global cutaneous oxygen saturation microcirculation at the plantar foot of healthy individuals was 8.4% higher than in patients with DM and 8.1% higher than in patients with PAOD (both P = 0.033). Patients with diabetes did not show significant differences in global cutaneous blood flow when compared with either healthy subjects or patients suffering from PAOD. CONCLUSIONS: Functional microcirculation at the plantar foot differs between healthy subjects and patients suffering from diabetes or PAOD of the same age. Patients with either diabetes or PAOD demonstrate deteriorated cutaneous oxygen saturation with equivalent blood perfusion at the plantar foot. More clinical studies have to be conducted to evaluate therapeutical methods that might ameliorate cutaneous oxygen saturation within diabetic foot disease and PAOD.

Compartmental and muscular response to closed soft tissue injury in rats investigated by oxygen-to-see and intravital fluorescence microscopy.

BACKGROUND: Closed soft tissue injury (CSTI) induces local inflammation and progressive microvascular dysfunction. The aim of the study was to evaluate and compare the microvascular changes systematically in a precompartmental tissue injury by oxygen-to-see (O2C), a combined laser Doppler flowmetry and spectrophotometry system, and intravital fluorescence microscopy (IVM). METHODS: Fourteen Wistar rats were subjected to a trauma and a control group (both n = 7). CSTI was performed on the left lower limb by means of a standardized impact device. Controls received a sham CSTI. Capillary blood flow (QRBC), oxygen saturation (sO2), and postcapillary filling pressure (rHb) were measured noninvasively by O2C assessed in 2-mm and 8-mm depth underneath the skin. Measurements were done before and after trauma and hourly up to 24 hours. IVM of the soleus muscle was performed after 24 hours. RESULTS: Before CSTI, O2C parameters did not reveal a difference between both groups. Up to 2 hours after trauma, QRBC was significantly increased in 8-mm tissue depth. No significant changes of sO2 and rHb were noted compared with controls. In 2-mm depth, significantly reduced QRBC and rHb levels were observed compared with 8 mm but with no significant changes after CSTI. IVM showed a significant increase of postcapillary blood flow with decreased functional capillary density, increased macromolecular leakage, and increased nicotinamide adenine dinucleotide hydride. CONCLUSIONS: After CSTI in rats, there was an immediate increase of compartmental capillary blood flow with a slight increase of muscle oxygen saturation and unchanged postcapillary venous filling pressures as sign of a redistribution of blood between soft and muscle tissue. The severity of pathologic changes in the compartment was not reflected by O2C but by IVM.

Reliability and consequences of intraoperative 3D imaging to control positions of thoracic pedicle screws.

INTRODUCTION: The insertion of thoracic pedicle screws (T1-T10) is subject to a relevant rate of malplacement. The optimum implantation procedure is still a topic of controversial debate. Currently, a postoperative computed tomography is required to evaluate the screw positions. The present study was undertaken to clarify whether intraoperative 3D imaging is a reliable method of determining the position of thoracic pedicle screws. METHODS: This prospective study involved 40 consecutive patients with thoracic spinal injuries, with intraoperative 3D scans being performed to determine the positions of 240 pedicle screws in T1-T10. The results of the 3D scans were compared with the findings of postoperative CT scans, using a clinical classification system. RESULTS: The positions of 204 pedicle screws could be viewed by means of both 3D and CT scans and the results compared. The 3D scans achieved a sensitivity of 90.9 % and a specificity of 98.8 %. The rate of misclassification by the 3D scans was 2.5 %. Nine pedicle screws were classified as misplaced and their position corrected intraoperatively (3.8 %). No screws required postoperative revision. CONCLUSIONS: Performing an intraoperative 3D scan enables the position of thoracic pedicle screws to be determined with sufficient accuracy. The rate of revision surgery was reduced to 0 %.

Erythropoietin enhances the regeneration of traumatized tissue after combined muscle-nerve injury.

BACKGROUND: Erythropoietin (EPO) is a pleiotropic cytokine with neuroprotective, anti-inflammatory, and muscle regenerative properties. The purpose of our study was to analyze the regenerative capacity of systemically applied EPO in a combined muscle-nerve injury model. METHODS: We performed a crush injury to the left soleus muscle in 84 male Wistar rats. Using an instrumented clamp, the muscle was crushed over its complete length. Simultaneously, the ipsilateral sciatic nerve was sham manipulated or crushed. Upon induction of the trauma, animals received either EPO (E) (single application of 5,000 IU/kg body weight intraperitonial) or vehicle solution (K). After in vivo assessment of mechanical pain according to Frey, thermal hyperalgesia, latency of nerve conduction velocity, and strength of the soleus muscle were analyzed at days 1, 7, and 42 postinjury (n = 7 per group). Cell proliferation and apoptosis were assessed by means of histology and immunohistochemistry. RESULTS: Combined muscle-nerve injury showed a significant loss of muscle strength, which incompletely recovered within 42 days. Rats treated with EPO showed an increased muscle strength after 7 days and 42 days compared with the control group. Pain behavior was highest in the muscle-nerve injured animals at day 7. EPO decreased the pain and increased nerve conduction velocity. Nerve injury diminished proliferation of muscle cells, whereas simultaneous therapy with EPO resulted in a boost of bromdesoxyuridine-positive cells. CONCLUSIONS: EPO promoted muscle restoration and enhanced nerve recovery after combined muscle-nerve injury. Thus, EPO might represent an attractive therapeutic option to optimize the posttraumatic course after injury.

Inhibition of caspase mediated apoptosis restores muscle function after crush injury in rat skeletal muscle.

Although muscle regeneration after injury is accompanied by apoptotic cell death, prolonged apoptosis inhibits muscle restoration. The goal of our study was to provide evidence that inhibition of apoptosis improves muscle function following blunt skeletal muscle injury. Therefore, 24 rats were used for induction of injury to the left soleus muscle using an instrumented clamp. All animals received either 3.3 mg/kg i.p. of the pan-caspase inhibitor Z-valinyl-alanyl-DL: -aspartyl-fluoromethylketone (z-VAD.fmk) (n = 12 animals) or equivalent volumes of the vehicle solution DMSO (n = 12 animals) at 0 and 48 h after trauma. After assessment of the fast twitch and tetanic contraction capacity of the muscle at days 4 and 14 post injury, sampling of muscle tissue served for analysis of cell apoptosis (cleaved caspase 3 immunohistochemistry), cell proliferation (BrdU immunohistochemistry) as well as of muscle tissue area and myofiber diameter (HE planimetric analysis). Muscle strength analysis after 14 days in the z-VAD.fmk treated group revealed a significant increase in relative muscle strength when compared to the DMSO treated group. In contrast to the DMSO treated injured muscle, showing a transient switch towards a fast-twitching muscle phenotype (significant increase of the twitch-to-tetanic force ratio), z-VAD.fmk treated animals showed an enhanced healing process with a faster restoration of the twitch-to-tetanic force ratio towards the physiological slow-twitching muscle phenotype. This enhancement of muscle function was accompanied by a significant decrease of cell apoptosis and cell proliferation at day 4 as well as by a significant increase of muscle tissue area at day 4. At day 14 after injury z-VAD.fmk treated animals presented with a significant increase of myofiber diameter compared to the DMSO treated animals. Thus, z-VAD.fmk could provide a promising option in the anti-apoptotic therapy of muscle injury.

Melatonin restores muscle regeneration and enhances muscle function after crush injury in rats.

The goal of this study was to provide evidence that melatonin improves muscle healing following blunt skeletal muscle injury. For this purpose, we used 56 rats and induced an open muscle injury. After injury, all animals received either daily melatonin or vehicle solution intraperitoneally. Subsequent observations were performed at day 1, 4, 7, and 14 after injury. After assessment of fast twitch and tetanic muscle force, we analyzed leukocyte infiltration, satellite cell number, and cell apoptosis. We further quantified the expression of the melatonin receptor and the activation of extracellular-signal-regulated kinase (ERK). Chronic treatment with melatonin significantly increased the twitch and tetanic force of the injured muscle at day 4, 7, and 14. At day 1, melatonin significantly reduced the leukocyte infiltration and significantly increased the number of satellite cells when compared to the control group. Consistent with this observation, melatonin significantly reduced the number of apoptotic cells at day 4. Furthermore, phosphorylation of ERK reached maximal values in the melatonin group at day 1 after injury. Additionally, we detected the MT1a receptor in the injured muscle and showed a significant up-regulation of the MT1a mRNA in the melatonin group at day 4. These data support the hypothesis that melatonin supports muscle restoration after muscle injury, inhibits apoptosis via modulation of apoptosis-associated signaling pathways, increases the number of satellite cells, and reduces inflammation.

Fibroblast growth factor-2-overexpressing myoblasts encapsulated in alginate spheres increase proliferation, reduce apoptosis, induce adipogenesis, and enhance regeneration following skeletal muscle injury in rats.

The fibroblast growth factor 2 (FGF-2) is known as pleiotropic cytokine with myoblast proliferative properties. In the present study, we tested the hypothesis that gene transfer of human FGF-2 via transplantation of genetically modified L8-myoblast encapsulated in alginate modulates the skeletal muscle recovery after crush injury in Wistar rats. Therefore, we performed a crush injury to the soleus muscle and transplanted alginate spheres containing myoblasts genetically modified to overexpress human FGF-2 (FGF-2) or a luciferase (LUC) cDNA at the site of injury. Animals that underwent muscle injury without transplantation of alginate spheres served as control (control). At day 4 after trauma the FGF-2 group showed significant higher mean values of cell proliferation (bromodeoxyuridine immunohistochemistry) and significant lower values of cell apoptosis (terminal deoxynucleotidyl transferase nick end labeling histology) compared to animals receiving luciferase-overexpressing myoblasts. At the same time point adiponectin expression (ACRP30 immunohistochemistry) was increased in the FGF-2 group exclusively. The p75(NTR) expression (p75(NTR) immunohistochemistry) significantly improved in both the FGF-2 and LUC group compared to the control group. Functional analysis of the injured muscle did not reveal a significant increase of the muscle force in the FGF-2 group compared to the control and LUC group 14 days after injury. In vitro analysis for 14 days of the FGF-2-modified spheres demonstrated at day 7 and day 14 a significant increase of the relative cell count as well as of the relative viable cell count in the FGF-2 myoblast spheres compared to luciferase myoblast spheres. Additionally, the expression of FGF-2 (enzyme-linked immunosorbent assay analysis) and luciferase (chemiluminescence analysis) persisted in vitro for 4 and 14 days, respectively. These results demonstrate that FGF-2-overexpressing myoblasts cannot considerably improve muscle strength but are able to modulate the proliferation as well as the apoptosis of injured muscle tissue mainly by conducting adipogenesis.