Direct comparison of the immunogenicity of major histocompatibility complex-I and -II deficient mesenchymal stem cells in vivo.

Mesenchymal stem cells (MSCs) play an important role in tissue engineering applications aiming at the regeneration or substitution of damaged tissues. In this context, off-the-shelf allogeneic MSCs would represent an attractive universal cell source. However, immune rejection is a major limitation for the clinical use of allogeneic MSCs. Immune rejection is mediated by the expression of major histocompatibility complexes (MHC)-I and -II on the donor cells. In this study, we eliminated MHC-I and/or MHC-II expression in human MSCs by using the CRISPR/Cas9 technology and investigated the effect of the individual or combined knockout of MHC-I and MHC-II on MSC survival after transplantation into immunocompetent mice. Elimination of MHC-I and/or MHC-II expression did not affect mesenchymal marker gene expression, viability, proliferation and the differentiation potential of MSCs in vitro. However, cell survival of transplanted MSCs was significantly elevated in MHC-I and MHC-II deficient MSCs. A direct side-by-side comparison does not reveal any significant difference in the immunogenicity of MHC-I and MHC-II knockout MSCs. Moreover, double knockout of MHC-I and MHC-II did not further increase in vivo cell survival of transplanted MSCs. Our results demonstrate that knockout of MHC-I and/or MHC-II represents an effective strategy to prevent immune rejection of allogeneic MSCs.

Collagen-Elastin and Collagen-Glycosaminoglycan Scaffolds Promote Distinct Patterns of Matrix Maturation and Axial Vascularization in Arteriovenous Loop-Based Soft Tissue Flaps.

INTRODUCTION: Autologous free flaps are the criterion standard for reconstructions of complex soft tissue defects; however, they are limited by donor-site morbidities. The arteriovenous (AV) loop model enables the generation of soft tissue constructs based on acellular dermal matrices with a functional microvasculature and minimal donor site morbidity. The ideal scaffold for AV loop-based tissue engineering has not been determined. METHODS: AV loops were placed into subcutaneous isolation chambers filled with either a collagen-elastin scaffold or a collagen-glycosaminoglycan scaffold in the thighs of rats. Matrix elasticity, neoangiogenesis, cell migration, and proliferation were compared after 14 and 28 days. RESULTS: Mean vessel count and area had increased in both matrices at 28 compared with 14 days. Collagen-elastin matrices showed a higher mean vessel count and area compared with collagen-glycosaminoglycan matrices at 14 days. At 28 days, a more homogeneous vascular network and higher cell counts were observed in collagen-elastin matrices. Collagen-glycosaminoglycan matrices, however, exhibited less volume loss at day 28. CONCLUSIONS: Collagen-based scaffolds are suitable for soft tissue engineering in conjunction with the AV loop technique. These scaffolds exhibit distinct patterns of angiogenesis, cell migration, and proliferation and may in the future serve as the basis of tissue-engineered free flaps as an individualized treatment concept for critical wounds.

Il10 and poly-dl-lactide-ɛ-caprolactone conduits in critical size nerve defect bridging-An experimental study.

INTRODUCTION: Aim of this study is to evaluate if regeneration in repair of nerve defects can be improved by combination of a poly-dl-lactide-ɛ-caprolactone conduit (PLC) with long-term release of anti-inflammatory Interleukin 10 (IL10), which is known to reduce intraneural scarring in nerve regeneration through its anti-inflammatoric properties. METHOD: Experiments were performed at 30 female Lewis rats. Conduits filled with fibrin (PLC-group n = 10) and fibrin loaded with IL10 (IL10-group n = 10) were compared to autologs nerve grafts (NG-group n = 10) in a 15 mm sciatic nerve gap lesion. Sciatic function index (SFI) and electrophysiological analyses were performed 16 weeks after surgery prior to histological evaluation. In histological analyses total nerve count, total nerve area, myelination index, and N-ratio were measured. Additionally, gastrocnemius muscle was weighed. RESULTS: SFI (NG-group:-50.68 ± 7.03%; PLC-group:-56.48  ± 2.30%; IL10-group:-56.54  ± 8.22%) and nerve conduction velocity (NG-group: 92.52  ± 4.64 m/s; PLC-group: 92.77  ± 5.07 m/s; IL10-group: 93.78  ±3.63 m/s) showed no significant differences after 16 weeks (P > 0.05). Significant higher axon count (17.592  ± 483) were observed in the NG-group compared to PLC- (6.722 ± 553) and IL10-group (6.842 ± 681) (P < 0.001). NG-group had significant highest nerve cross sections (604.214  ± ±15.217 µm2 ) as compared to PLC- (245.669  ± ±28.034 µm2 ) and IL10-group (244.698 ± 26.772 µm2 ) (P < 0.001). Comparison of myelination index showed significant higher values for NG-group (0.46  ± 0.02) than PLC- (0.64  ± 0.01) and IL10-group (0.62  ± 0.01) (P < 0.001). N-ratios in PLC-group (0.21  ± 0.01) and IL10-group (0.24 ± 0.01) were lower than in NG-group (0.51  ± 0.03) (P < 0.001). Between PLC- and IL10-group no differences were observed (P > 0.05). Gastrocnemius muscle was heavier in NG-group (0.86 ± 0.21g) as compared to PLC- (0.26 ± 0.05g) and IL-10 group (0.29 ± 0.06 g) (P < 0.05). CONCLUSION: Bridging critical nerve defects through fibrin-filled PLC conduits is possible. Although, autologs nerve graft showed superior histological results. Long-term release of IL10 in the conduit did not improve regeneration of critical nerve defects. © 2015 Wiley Periodicals, Inc. Microsurgery 36:410-416, 2016.

C3 toxin and poly-DL-lactide-ε-caprolactone conduits in the critically damaged peripheral nervous system: a combined therapeutic approach.

INTRODUCTION: Peripheral nerve regeneration over longer distances through conduits is limited. In the presented study, critical size nerve gap bridging with a poly-DL-lactide-ε-caprolactone (PLC) conduit was combined with application of C3 toxin to facilitate axonal sprouting. MATERIALS AND METHODS: The PLC filled with fibrin (n = 10) and fibrin gel loaded with 1-µg C3-C2I and 2-µg C2II (n = 10) were compared to autologous nerve grafts (n = 10) in a 15-mm sciatic nerve gap lesion model of the rat. Functional and electrophysiological analyses were performed before histological evaluation. RESULTS: Evaluation of motor function and nerve conduction velocity at 16 weeks revealed no differences between the groups. All histological parameters and muscle weight were significantly elevated in nerve graft group. No differences were observed in both PLC groups. CONCLUSIONS: The PLCs are permissive for nerve regeneration over a 15-mm defect in rats. Intraluminal application of C3 toxin did not lead to significant enhancement of nerve sprouting.

An Analysis of the Implantable Doppler Probe for Postoperative Free-Flap Monitoring and Risk Factor Analysis for Revision Surgery in Facial Reanimation Surgery.

Background: Postoperative monitoring after free functional gracilis transfer for smile reconstruction in long-standing facial paralysis is challenging as clinical assessments are limited. Objective: In patients receiving free gracilis transfer for smile reconstruction, we compared the implantable Doppler probe with a handheld Doppler/intraoperative blood flow regarding the reliability in detecting perfusion compromised free flaps. Methods: In a retrospective cohort study we analyzed facial paralysis patients who, after free functional smile reconstruction, were postoperatively monitored using the implantable Doppler probe. Furthermore, we conducted a multiple logistic regression analysis on risk factors for vascular complications. Results: We included 119 patients who received 125 free functional gracilis transfers. The sensitivity of the implanted Doppler probe was 1.0 and the specificity 0.88. There were no false-negative results (negative predictive value = 1.0). The calculated positive predictive value was 0.41. We used a handheld Doppler device to verify signal changes. The combined positive predictive value of both tests was 0.91. Previous surgery in the surgical field was a risk factor for impaired blood flow. Conclusions: The implantable Doppler probe proved to be a reliable tool for postoperative monitoring of free functional gracilis transfer in facial reanimation surgery. Special care should be taken in preoperated patients.

Risk factors in distal interphalangeal joint arthrodesis in the hand: a retrospective study of 173 cases.

In this retrospective study we aimed to analyse the risk factors for complications after different methods of distal interphalangeal arthrodesis in the hand. Forty-four per cent were treated with K-wire/cerclage fixation, 46% with X-fuse® implants (Stryker GmbH, Selzach, Switzerland) and 10% with headless compression screws (HBS®-screw, KLS Martin Group, Tuttlingen, Germany). The median follow-up was 16 weeks (range 6-224). The overall complication incidence was 44% (minor complications 29% and major 15%). The logistic regression showed that osteoarthritis, revisional arthrodesis and smoking had a negative impact on the total complication incidence. A Cox-regression analysis showed that HBS®-screw arthrodesis was associated with a significantly lower incidence of major complications compared with K-wire/cerclage and X-Fuse®-arthrodesis. Revisional arthrodesis was five times more frequently connected with major complications than primary surgery. Smokers were three times more likely to experience major complications than non-smokers. We conclude that arthrodesis of the distal interphalangeal joint often leads to complications. Risk factors must be kept in mind.Level of evidence: III.

Interleukin-10 and regeneration in an end-to-side nerve repair model of the rat.

End-to-side (ETS) neurorrhaphy is an option in peripheral nerve surgery. The aim of this study was to investigate whether the application of the anti-inflammatory cytokine interleukin-10 (IL-10) reduces scarring and thus enhances nerve regeneration in an ETS peroneal/tibial nerve lesion model of the rat. Twenty rats with a peroneal to tibial ETS neurorrhaphy were divided into two groups: (1) control group and (2) IL-10 group with intrafascicular application of 0.125 µg/100 µl IL-10. Survival time was 8 weeks. Nerve conduction velocities (NCVs) and motor function were analyzed and histomorphological evaluation with measurement of intraneural collagen level, axon count, total nerve area, and myelination index followed. Evaluation of motor function and nerve conduction did not show any statistical differences. Histological analyses revealed thicker myelin sheaths and higher myelination index in the IL-10 group (p < 0.001). Axon count showed no difference. The IL-10 group revealed lower collagen levels (p < 0.001). Comparison of total nerve area showed no statistical significance. At this dose, IL-10 evaluated at 8 weeks was not significantly different than placebo in functional, NCVs, and most morphological measures. However, there was a significant difference in thicker myelin sheaths and higher myelination index and lower collagen levels. This suggests that future experiments of IL-10 at different doses or longer periods of evaluation would be of interest.

Ulnocarpal Impaction.

Ulnocarpal impaction syndrome is a common cause for ulnarsided wrist pain caused by an abutment between the ulnar head and the lunotriquetral complex. This pain is typically triggered by load bearing and rotation of the forearm. Radiographic examination is often associated with positive ulnar variance and cysts in the lunate, edema of the ulnoproximal lunate is shown in MRI. Operative treatment aims to reduce load on the lunate, either by open ulnar shortening osteotomy or arthroscopic wafer procedure.

Revisionary soft tissue reconstruction of posterior midline defects after spinal surgery-plastic reconstructive options including perforator flaps.

BACKGROUND: Chronic prevertebral soft tissue defects with exposed metal hardware following spinal surgery represent a challenging complication. Frequently patients underwent multiple previous operations due to wound complications. Surrounding soft tissues are often compromised due to malperfusion, severe subcutaneous scarring, previous local advancement flaps and therefore impair stable wound closure. METHODS: Patients after spinal surgery who received complex soft tissue reconstructions between 2011 and 2015 were analyzed retrospectively. Patient`s age, risk factors, wound size, cause and defect location as well as complication rates were evaluated. A focus was set on therapeutic strategies and decision-making concerning reconstructive techniques. RESULTS: Fourteen patients receiving 27 pedicled and one free flap were included in the study. Patients mean age was 51.1 years, mean wound size was 144 cm2. Defects were located in the lumbar spine [8], cervical spine [2] and thoracic spine [1], respectively. Three patients suffered from extensive defects affecting more than one area. Mean time of flap surgery was 213 minutes. Fifteen perforator-based flaps and 11 non-perforator (classic rotation-flaps), 1 pedicled and 1 free latissimus dorsi flap were used. In 9 patients (64.3%) different flaps had to be combined in a single-staged procedure due to large wounds. Implant material was removed completely in six patients (42.9%), whereas in five patients (35.7%) implants were replaced within the operation for soft tissue reconstruction. In three patients (21.4%) initial implant removal or replacement was not possible which leads to prolonged postoperative wound infections. CONCLUSIONS: Most patients with exposed spinal hardware suffered from multiple comorbidities and showed a poor general condition. Due to the reduced soft tissue quality wound healing is significantly impaired. Exposed implant material should be replaced or removed when possible. Therefore, the complete armamentarium of plastic reconstructive techniques is required for wound closure. Today, perforator flaps play a prominent role due to the variability, excellent vascularization and sufficient subcutaneous filling capacities.

Haemodynamically stimulated and in vivo generated axially vascularized soft-tissue free flaps for closure of complex defects: Evaluation in a small animal model.

The arteriovenous (AV) loop model permits the creation of significant volumes of axially vascularized tissue that represents an alternative to conventional free flaps, circumventing their common limitations. However, such AV loop-based flaps have never before been examined in standardized animal models with respect to their suitability for reconstruction of critical bone-exposing defects. In the course of our preliminary studies, we implemented a novel defect model in rats that provides standardized and critical wound conditions and evaluated whether AV loop-generated flaps are suitable for free microsurgical transfer and closure of composite defects. We compared three groups of rodents with similar scapular defects: one received the AV flap, whereas controls were left to heal by secondary intention or with supplementary acellular matrix alone. To create the flaps, AV loops were placed into subcutaneous Teflon chambers filled with acellular matrix and transferred to the thigh region. Flap maturation was evaluated by histological analysis of angiogenesis and cell migration at days 14 and 28 after loop creation. Flap transfer to the scapular region and microsurgical anastomoses were performed after 14 days. Postoperative defect closure and perfusion were continually compared between groups. Within the AV flap chamber, the mean vessel number, cell count and the proportion of proliferating cells increased significantly over time. The novel defect model revealed that stable wound coverage with homogeneous vascular integration was achieved by AV loop-vascularized soft-tissue free flaps compared with controls. In summary, our study indicates for the first time that complex composite defects in rats can successfully be treated with AV loop-based free flaps.

Flow-Induced Axial Vascularization: The Arteriovenous Loop in Angiogenesis and Tissue Engineering.

Fabrication of a viable vascular network providing oxygen supply is identified as one crucial limiting factor to generate more complex three-dimensional constructs. The arteriovenous loop model provides initial blood supply and has a high angioinductive potency, making it suitable for vascularization of larger, tissue-engineered constructs. Also because of its angiogenic capabilities the arteriovenous loop is recently also used as a model to evaluate angiogenesis in vivo. This review summarizes the history of the arteriovenous loop model in research and its technical and surgical aspects. Through modifications of the isolation chamber and its containing matrices, tissue generation can be enhanced. In addition, matrices can be used as release systems for local application of growth factors, such as vascular endothelial growth factor and basic fibroblast growth factor, to affect vascular network formation. A special focus in this review is set on the assessment of angiogenesis in the arteriovenous loop model. This model provides good conditions for assessment of angiogenesis with the initial cell-free environment of the isolation chamber, which is vascularized by the arteriovenous loop. Because of the angiogenic capabilities of the arteriovenous loop model, different attempts were performed to create functional tissue in the isolation chamber for potential clinical application. Arteriovenous loops in combination with autologous bone marrow aspirate were already used to reconstruct large bone defects in humans.

Flow increase is decisive to initiate angiogenesis in veins exposed to altered hemodynamics.

Exposing a vein to altered hemodynamics by creating an arteriovenous (AV) shunt evokes considerable vessel formation that may be of therapeutic potential. However, it is unclear whether the introduction of oscillatory flow and/or flow increase is decisive. To distinguish between these mechanical stimuli we grafted a femoral vein into the arterial flow pathway of the contralateral limb in rats creating an arterioarterial (AA) loop (n = 7). Alternatively, we connected the femoral artery and vein using the vein graft, whereby we created an AV-loop (n = 27). Vessel loops were embedded in a fibrin filled chamber and blood flow was measured by means of flow probes immediately after surgery (day 0) and 15 days after loop creation. On day 15, animals were sacrificed and angiogenesis was evaluated using µCT and histological analysis. Mean flow increased from 0.5 to 2.4 mL/min and was elevated throughout the cardiac cycle at day 0 in AV-loops whereas, as expected, it remained unchanged in AA-loops. Flow in AV-loops decreased with time, and was at day 15 not different from untreated femoral vessels or AA-loop grafts. Pulsatile flow oscillations were similar in AV-and AA-loops at day 0. The flow amplitude amounted to ~1.3 mL/min which was comparable to values in untreated arteries. Flow amplitude remained constant in AA-loops, whereas it decreased in AV-loops (day 15: 0.4 mL/min). A large number of newly formed vessels were present in AV-loops at day 15 arising from the grafted vein. In marked contrast, angiogenesis originating from the grafted vein was absent in AA-loops. We conclude that exposure to substantially increased flow is required to initiate angiogenesis in grafted veins, whereas selective enhancement of pulsatile flow is unable to do so. This suggests that indeed flow and most likely wall shear stress is decisive to initiate formation of vessels in this hemodynamically driven angiogenesis model.

Necrotizing fasciitis after central venous catheter placement.

BACKGROUND: Necrotizing fasciitis (NF) can appear after various penetrating or non-penetrating skin lesions. This is the first reported case in which NF occurred after a central venous line placement. Because of intubation and sedation of the critically ill patient, only local conditions can indicate NF although other decisive symptoms, such as pain out of proportion to physical findings, are not evaluable. METHODS: Case report and review of the literature. RESULTS: A 71-y-old male patient was admitted to the intensive care unit after spine surgery due to post-operative delirium. Because of respiratory failure he had to be intubated and sedated. Ten days after central venous line placement in the right subclavian vein a reddening and swelling of the insertion site was observed but considered as extravasation. When aggravation of the local symptoms occurred, the suspected diagnosis of NF was made and a radical debridement was performed immediately. After a second-look operation, defect closure with a free-flap transfer and split-thickness skin grafting could be achieved. CONCLUSIONS: The first report on NF in a critically ill patient due to a subclavian central intravenous line aims to encourage checking for iatrogenic soft tissue condition in sedated intensive care patients. These patients may have a greater risk of developing NF, because they often have predisposing factors such as diabetes, end-stage renal failure, and immune suppression.