Tobias Winkler

Dose-response relationship of mesenchymal stem cell transplantation and functional regeneration after severe skeletal muscle injury in rats.

Various therapeutic strategies that aim to influence clinical outcome after severe skeletal muscle trauma have been considered. One such method, the local transplantation of stem cells, has been shown to improve tissue regeneration. The number of cells required for successful regeneration, however, remains unclear. The aim of this study was therefore to examine the correlation between the number of transplanted bone marrow-derived mesenchymal stem cells (MSCs) and the resulting muscle function. One week after inducing an open crush trauma in 34 female Sprague Dawley rats, increasing quantities of autologous MSCs (0.1 x 10(6), 1 x 10(6), 2.5 x 10(6), and 10 x 10(6) cells) or saline solution (control group) were transplanted into the left soleus muscle of the rat hind limb. At 4 weeks posttrauma, the outcome was assessed by measuring muscle contraction forces following an indirect fast twitch and tetanic stimulation. A logarithmic dose-response relationship was observed for both maximum twitch and tetanic contraction forces (R(2) = 0.9 for fast twitch [p = 0.004]; R(2) = 0.87 [p = 0.002] for tetanic contraction). The transplantation of 10 x 10(6) cells resulted in the most pronounced improvement of muscle force. MSC therapy represents a promising new tool for the treatment of skeletal muscle trauma that shows potential for aiding in the prevention of severe functional deficiencies. The logarithmic dose-response relationship demonstrates the association between the number of transplanted cells and the resulting muscle forces, as well as the amount of MSCs required for promoting muscular regeneration.

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.

Time course of skeletal muscle regeneration after severe trauma

Background and purpose Animal models of skeletal muscle injury should be thoroughly described and should mimic the clinical situation. We established a model of a critical size crush injury of the soleus muscle in rats. The aim was to describe the time course of skeletal muscle regeneration using mechanical, histological, and magnetic resonance (MR) tomographic methods. Methods Left soleus muscles of 36 Sprague-Dawley rats were crushed in situ in a standardized manner. We scanned the lower legs of 6 animals by 7-tesla MR one week, 4 weeks, and 8 weeks after trauma. Regeneration was evaluated at these times by in vivo measurement of muscle contraction forces after fast-twitch and tetanic stimulation (groups 1W, 4W, 8W; 6 per group). Histological and immunohistological analysis was performed and the amount of fibrosis within the injured muscles was determined histomorphologically. Results MR signals of the traumatized soleus muscles showed a clear time course concerning microstructure and T1 and T2 signal intensity. Newly developed neural endplates and myotendinous junctions could be seen in the injured zones of the soleus. Tetanic force increased continuously, starting at 23% (SD 4) of the control side (p < 0.001) 1 week after trauma and recovering to 55% (SD 23) after 8 weeks. Fibrotic tissue occupied 40% (SD 4) of the traumatized muscles after the first week, decreased to approximately 25% after 4 weeks, and remained at this value until 8 weeks. Interpretation At both the functional level and the morphological level, skeletal muscle regeneration follows a distinct time course. Our trauma model allows investigation of muscle regeneration after a standardized injury to muscle fibers.

Synthetic niche to modulate regenerative potential of MSCs and enhance skeletal muscle regeneration

Severe injury to the skeletal muscle often results in the formation of scar tissue, leading to a decline in functional performance. Traditionally, tissue engineering strategies for muscle repair have focused on substrates that promote myogenic differentiation of transplanted cells. In the current study, the reported data indicates that mesenchymal stromal cells (MSCs) transplanted via porous alginate cryogels promote muscle regeneration by secreting bioactive factors that profoundly influence the function of muscle progenitor cells. These cellular functions, which include heightened resistance of muscle progenitor cells to apoptosis, migration to site of injury, and prevention of premature differentiation are highly desirable in the healing cascade after acute muscle trauma. Furthermore, stimulation of MSCs with recombinant growth factors IGF-1 and VEGF165 was found to significantly enhance their paracrine effects on muscle progenitor cells. Multifunctional alginate cryogels were then utilized as synthetic niches that facilitate local stimulation of seeded MSCs by providing a sustained release of growth factors. In a clinically relevant injury model, the modulation of MSC paracrine signaling via engineered niches significantly improved muscle function by remodeling scar tissue and promoting the formation of new myofibers, outperforming standalone cell or growth factor delivery.

Fiber type characterization in skeletal muscle by diffusion tensor imaging.

Fiber type distribution within a skeletal muscle, i.e. the quantification of the relative amount of type 1 (slow‐twitching) and type 2 (fast‐twitching) muscle fibers, is of great interest for the monitoring of the effects of training or the treatment of muscle diseases. The purpose of this study was to determine the feasibility of diffusion tensor imaging (DTI) as a tool for noninvasive fiber type quantification in human skeletal muscle. The right calves of 12 healthy volunteers were examined using DTI at 1.5 T. Standard DTI parameters, including fractional anisotropy (FA), and mean, radial and parallel diffusivity (MD, RD and PD, respectively), were determined in the soleus muscle. Fiber type proportion and mean fiber diameter within the soleus muscle were quantified from tissue specimens obtained via a fine needle biopsy. Linear regression analysis tested for associations between DTI and biopsy results. FA values were correlated significantly with fiber type proportion, such that higher FA values indicated a higher proportion of type 1 fibers (R2 = 0.5, p = 0.01). This was based on lower diffusivity perpendicular to the main axis of the fiber in subjects with a higher type 1 fiber proportion (RD: R2 = 0.52, p = 0.008). MD was also correlated with the proportion of type 1 fibers (R2 = 0.37, p = 0.037), whereas PD showed no significant correlation. DTI is a promising method for the noninvasive estimation of fiber type proportion in skeletal muscle. This technique may be used to monitor training effects or may be further developed as a biomarker in certain muscle diseases. Copyright

Interscalene versus subacromial continuous infusion of ropivacaine after arthroscopic acromioplasty: a randomized controlled trial.

INTRODUCTION Interscalene and subacromial infusion of local anesthetics have both been shown to be effective in alleviating pain after shoulder arthroscopy. We performed a prospective randomized clinical trial in which both methods were compared in patients after acromioplasty. METHODS Forty patients received a subacromial (n = 20) or an interscalene (n = 20) continuous infusion of 2% ropivacaine (2 mL/h) after arthroscopic acromioplasty. Visual analog scale pain scores, additional medication requirements, and side effects were evaluated for 43 hours. The incidence of night pain was recorded. RESULTS Compared with the subacromial infusion, the continuous interscalene infusion of ropivacaine in the operated-on shoulder resulted in significantly reduced visual analog scale pain scores measured during rest, and also during exercise at 8 and 12 hours after surgery. The incidence of night pain was significantly lower in the interscalene group (P = .018). CONCLUSION A continuous subacromial infusion of 0.2% ropivacaine (2 mL/h) is inferior to continuous interscalene infusion, particularly during the first 12 hours, but could be an alternative in patients with contraindications of interscalene anesthesia or when acromioplasty is performed as an outpatient procedure.

A formula to predict patients' gluteus medius muscle volume from hip joint geometry.

The volume of the gluteus medius muscle (GMV) has been shown to be closely related to hip joint function. A defined relation between joint geometry and GMV would allow a calculation of the patient-specific GMV providing reference values to individually determine the goals for rehabilitation programs after total hip arthroplasty (THA). The aim of this study was to investigate correlations between hip geometry and GMV. One hundred and two (50 female, age: 58.53 (18-86)) pelvic computed tomography (CT) scans were analyzed to determine femoral offset (FO), body weight lever arm (BWLA) and the GMV. Relationships between demographic data, FO and GMV were analyzed using correlation and regression analysis. The mean GMV was found to be 289 ± 72 cm(3), the mean FO measured was 4.14 ± 0.55 cm; and the mean value for BWLA measured was 8.88 ± 0.4 cm. A formula to calculate the GMV with a good coefficient of determination (R(2) = 0.681) (p < 0.0001) was derived. In conclusion, the formula obtained predicts individual GMV with good model fit and could be used to individually determine rehabilitation goals. Moreover, the correlation found could account for a hand in hand development of FO and GMV during growth and a continuous functional relationship thereafter.

Biomechanical, microvascular, and cellular factors promote muscle and bone regeneration

It is becoming clear that the long-term outcome of complex bone injuries benefits from approaches that selectively target biomechanical, vascular, and cellular pathways. The typically held view of either biological or mechanical aspects of healing is oversimplified and does not correspond to clinical reality. The fundamental mechanisms of soft tissue regeneration most likely hold the key to understanding healing response.

Immediate and delayed transplantation of mesenchymal stem cells improve muscle force after skeletal muscle injury in rats

Mesenchymal stem cell (MSC) therapy is a promising approach for regaining muscle function after trauma. Prior to clinical application, the ideal time of transplantation has to be determined. We investigated the effects of immediate and delayed transplantation. Sprague–Dawley rats received a crush trauma to the left soleus muscle. Treatment groups were transplanted locally with 2 × 106 autologous MSCs, either immediately or 7 days after trauma. Saline was used as sham therapy. Contraction force tests and histological analyses were performed 4 weeks after injury. GFP‐labelled MSCs were followed after transplantation. The traumatized soleus muscles of the sham group displayed a reduction of twitch forces to 36 ± 17% and of tetanic forces to 29 ± 11% of the non‐injured right control side, respectively. Delayed MSC transplantation resulted in a significant improvement of contraction maxima in both stimulation modes (twitch, p = 0.011; tetany, p = 0.014). Immediate transplantation showed a significant increase in twitch forces to 59 ± 17% (p = 0.043). There was no significant difference in contraction forces between muscles treated by immediate and delayed cell transplantation. We were able to identify MSCs in the interstitium of the injured muscles up to 4 weeks after transplantation. Despite the fundamental differences of the local environment, which MSCs encounter after transplantation, similar results could be obtained with respect to functional muscle regeneration. We believe that transplanted MSCs residing in the interstitial compartment evolve their regenerative capabilities through paracrine pathways. Our data suggest a large time window of the therapeutical measures. Copyright

Preoperative irradiation for the prevention of heterotopic ossification induces local inflammation in humans

Radiation of the hip is an established method to prevent heterotopic ossification (HO) following total hip arthroplasty (THA) but the precise mechanism is unclear. As inflammatory processes are suggested to be involved in the pathogenesis of HO, we hypothesized that the preoperative irradiation impacts local immune components. Therefore, we quantified immune cell populations and cytokines in hematomas resulting from the transection of the femur in two groups of patients receiving THA: patients irradiated preoperatively (THA-X-hematoma: THA-X-H group) in the hip region (7 Gy) in order to prevent HO and patients who were not irradiated (THA-H group) but were postoperatively treated with non-steroidal anti-inflammatory drugs (NSAIDs). Radiation resulted in significantly increased frequencies of T cells, cytotoxic T cells, NKT cells and CD25+CD127- Treg cells, whereas the number of naive CD45RA-expressing cytotoxic T cells was reduced. These results indicate differential immune cell activation, corroborated by our findings of significantly higher concentrations of pro-inflammatory cytokines (e.g., IL-6, IFNγ) and chemokines (e.g., MCP-1, RANTES) in the THA-X-H group as compared to THA-H group. In contrast, the concentration of the angiogenic VEGF was significantly suppressed in the THA-X-H group. We conclude that preoperative irradiation results in significant changes in immune cell composition and cytokine secretion in THA-hematomas, establishing a specific - rather proinflammatory - milieu. This increase of inflammatory activity together with the observed suppression in VEGF secretion may contribute to the prevention of HO.