Robert Rotter

Erythropoietin Improves Functional and Histological Recovery of Traumatized Skeletal Muscle Tissue

Apart from its hematopoietic effect, erythropoietin (EPO) is known as pleiotropic cytokine with anti‐inflammatory and anti‐apoptotic properties. Here, we evaluated for the first time the EPO‐dependent regeneration capacity in an in vivo rat model of skeletal muscle trauma. A myoblast cell line was used to study the effect of EPO on serum deprivation‐induced cell apoptosis in vitro. A crush injury was performed to the left soleus muscle in 80 rats treated with either EPO or saline. Muscle recovery was assessed by analysis of contraction capacities. Intravital microscopy, BrdU/laminin double immunohistochemistry and cleaved caspase‐3 immunohistochemistry of muscle tissue on days 1, 7, 14, and 42 posttrauma served for assessment of local microcirculation, tissue integrity, and cell proliferation. Serum deprivation‐induced myoblast apoptosis of 23.9 ± 1.5% was reduced by EPO to 17.2 ± 0.8%. Contraction force analysis in the EPO‐treated animals revealed significantly improved muscle strength with 10–20% higher values of twitch and tetanic forces over the 42‐day observation period. EPO‐treated muscle tissue displayed improved functional capillary density as well as reduced leukocytic response and consecutively macromolecular leakage over day 14. Concomitantly, muscle histology showed significantly increased numbers of BrdU‐positive satellite cells and interstitial cells as well as slightly lower counts of cleaved caspase‐3‐positive interstitial cells. EPO results in faster and better regeneration of skeletal muscle tissue after severe trauma and goes along with improved microcirculation. Thus, EPO, a compound established as clinically safe, may represent a promising therapeutic option to optimize the posttraumatic course of muscle tissue healing.

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-β 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-β 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.

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

Abstract:  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.

Biomechanical in vitro testing of human osteoporotic lumbar vertebrae following prophylactic kyphoplasty with different candidate materials.

Study Design. Biomechanical study of human osteoporotic lumbar vertebrae following prophylactic kyphoplasty. Objectives. To evaluate the potential benefits of different resorbable candidate materials for use in prophylactic kyphoplasty compared with the behavior of polymethylmethacrylate cement. Summary of Background Data. Kyphoplasty using PMMA bone cement for the stabilization of fractured osteoporotic vertebrae has been established as a useful clinical tool. In several studies, consecutive compression fractures have been reported in vertebrae caudal or cranial to those augmented with bone cement. Consequently, some physicians have begun to treat adjacent vertebrae by means of prophylactic augmentation. Methods. Biomechanical in vitro testing was performed on 40 human osteoporotic nonfractured lumbar vertebrae. Three types of bone cement (PMMA, 2 different calcium phosphate cements) and one silicon derivative were assessed during compressive and cyclic sinusoidal testing. Each candidate material was applied bipedicularly under fluoroscopic control. Results. Differing processing qualities of the materials led to substantial differences during cement injection, in particular in the amount of cement filling of the vertebrae. However, in comparison to native vertebrae, augmented specimens showed significantly higher compressive failure. No significant differences between vertebral bodies treated with PMMA and those treated with either type of calcium phosphate cement were documented. The biomechanical properties of the vertebrae could not be significantly improved by the silicon derivative. Conclusion. This study demonstrated that calcium phosphate cements displayed identical behavior to PMMA cement with respect to in vitro mechanical qualities. Consequently, from a mechanical viewpoint, calcium phosphate cements may be used in addition to PMMA cement for kyphoplasty and prophylactic kyphoplasty. Silicon derivatives are apparently not recommendable as candidate materials for kyphoplasty.

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.

Open blunt crush injury of different severity determines nature and extent of local tissue regeneration and repair.

Insufficiency of skeletal muscle regeneration is often accompanied with functional deficiencies. The goal of our study was to assess the restoration of peripheral muscle upon injury of different severity. Blunt crush injury of the soleus muscle in rats was induced by a clamp and stepwise amplified in severity by rising the locking level of the clamp, resulting in three different groups (1× lock; 2× lock; 3× lock; n = 30 animals per group). After assessment of the fast twitch and tetanic contraction capacity at days 1, 4, 7, 14, and 42 postinjury sampling of muscle tissue served for analysis of cell proliferation, including satellite cells, apoptosis, and leukocyte infiltration. Contraction force analysis demonstrated significantly higher values of relative muscle strength in the 1× lock group compared to the two other groups over 42 days. Calculation of the twitch‐to‐tetanic force ratio revealed significantly higher mean values at days 1, 7, and 14 in the animals of group 2× lock and 3× lock, indicating a transformation toward a fast‐twitching muscular phenotype. Moreover, cell proliferation during the first 4 days was found dependent on the severity of muscle injury in that the higher the severity the higher the proliferation. At the same time, cell apoptosis was found increased, and at day 1 the local leukocyte infiltration was significantly higher in the 3× lock compared to the 1× lock group. These data indicate that severity of injury correlates with local repair responses, which, however, are not necessarily sufficient to fully restore muscle function.

Achilles tendon suture deteriorates tendon capillary blood flow with sustained tissue oxygen saturation – an animal study

BackgroundTreatment of ruptured Achilles tendons currently constitutes of conservative early functional treatment or surgical treatment either by open or minimal invasive techniques. We hypothesize that an experimental Achilles tendon suture in an animal model significantly deteriorates Achilles tendon microcirculation immediately following suturing.MethodsFifteen Achilles tendons of eight male Wistar rats (275–325 g) were included. After preparation of the Achilles tendon with a medial paratendinous approach, Achilles tendon microcirculation was assessed using combined Laser-Doppler and spectrophotometry (Oxygen-to-see) regarding:- tendinous capillary blood flow [arbitrary units AU]- tendinous tissue oxygen saturation [%]- tendinous venous filling pressure [rAU]The main body of the Achilles tendon was measured in the center of the suture with 50 Hz. 10 minutes after Achilles tendon suture (6-0 Prolene), a second assessment of microcirculatory parameters was performed.ResultsAchilles tendon capillary blood flow decreased by 57% following the suture (70 ± 30 AU vs. 31 ± 16 AU; p < 0.001). Tendinous tissue oxygen saturation remained at the same level before and after suture (78 ± 17% vs. 77 ± 22%; p = 0.904). Tendinous venous filling pressure increased by 33% (54 ± 16 AU vs. 72 ± 20 AU; p = 0.019) after suture.ConclusionAchilles tendon suture in anaesthetised rats causes an acute loss of capillary perfusion and increases postcapillary venous filling pressures indicating venous stasis. The primary hypothesis of this study was confirmed. In contrast, tendinous tissue oxygen saturation remains unchanged excluding acute intratendinous hypoxia within the first 10 minutes after suture. Further changes of oxygen saturation remain unclear. Furthermore, it remains to be determined to what extent reduced capillary blood flow as well as increased postcapillary stasis might influence tendon healing from a microcirculatory point of view in this animal setting.

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.

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.

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/cm2;), tissue hypoxia (nicotinamide adenine dinucleotide autofluorescence: 77±4 aU), and enhanced leukocyte-endothelial cell interaction (773±35 cells/mm2;). Therapeutic intervention with antithrombin 6 hrs after trauma restored nutritive perfusion and tissue oxygenation (functional capillary density: 469±22cm/cm2; nicotinamide adenine dinucleotide autofluorescence: 61±5 aU [p < 0.05]) and reduced inflammatory leukocyte adherence (237±20 cells/mm2; [p < 0.05]) toward values found in nontraumatized controls (functional capillary density: 573±13cm/cm2; nicotinamide adenine dinucleotide autofluorescence: 56±2 aU; leukocyte adherence: 204±20 cells/mm2;). 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.