The angiogenesis inhibitor vasostatin is regulated by neutrophil elastase-dependent cleavage of calreticulin in AML patients

The calcium-binding protein calreticulin (CRT) regulates protein folding in the endoplasmic reticulum (ER) and is induced in acute myeloid leukemia (AML) cells with activation of the unfolded protein response. Intracellular CRT translocation to the cell surface induces immunogenic cell death, suggesting a role in tumor suppression. In this study, we investigated CRT regulation in the serum of patients with AML. We found that CRT is not only exposed by exocytosis on the outer cell membrane after treatment with anthracyclin but also ultimately released to the serum in vitro and in AML patients during induction therapy. Leukemic cells of 113 AML patients showed increased levels of cell-surface CRT (P < .0001) and N-terminus serum CRT (P < .0001) compared with normal myeloid cells. Neutrophil elastase was identified to cleave an N-terminus CRT peptide, which was characterized as vasostatin and blocked ATRA-triggered differentiation. Levels of serum vasostatin in patients with AML inversely correlated with bone marrow vascularization, suggesting a role in antiangiogenesis. Finally, patients with increased vasostatin levels had longer relapse-free survival (P = .04) and specifically benefited from autologous transplantation (P = .006). Our data indicate that vasostatin is released from cell-surface CRT and impairs differentiation of myeloid cells and vascularization of the bone marrow microenvironment.

Autotransplant for Hodgkin lymphoma after failure of upfront BEACOPP escalated (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone)

BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) escalated is the preferred upfront Hodgkin lymphoma (HL) treatment in a number of countries. Upon failure, high-dose chemotherapy with autologous stem cell support (HDT/ASCT) is performed, but its effectiveness has not been verified in this setting. We analyzed all Swiss cases of chemosensitive HL autografted after failure of BEACOPP escalated (n = 22) and compared outcomes with 22 cases of HDT/ASCT following frontline ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) failure. Five-year progression-free survival (PFS) was 76% for ABVD and 42% for BEACOPP escalated (p = 0.029). Two- and 5-year overall survival (OS) was 90% and 71% for ABVD and 72% and 65% for BEACOPP escalated, respectively (p = not significant). Three patients in the ABVD and four in the BEACOPP escalated groups underwent allotransplant for relapse after HDT/ASCT. Grade 3-4 toxicities were comparable in both groups. Three cases of therapy-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/t-AML) were recorded in the BEACOPP escalated group. The acceptable PFS and OS of chemosensitive patients with HL autografted after failure of upfront BEACOPP escalated seem to justify this approach.

Microsurgical management of large intra-and extracranial calvarial meningiomas

Objective: Based on the largest series reported of giant intra- and extracranial calvarial meningiomas (GIECM) the purpose of the present study was to characterize the treatment and outcome data associated with patients operated on GIECM and to describe our experience in the management of this rare and therapeutically demanding tumour entity. Methods: The data of 12 patients (7/12 males, 5/12 females) with surgically treated GIECM at the University Hospitals Aachen and Bern between 1994 and 2011 were retrospectively analyzed. The mean patient age was 58 years (range, 22 to 78 years). Symptom distribution included extracranial swelling (12/12), seizures (5/12), headache (4/12), gait disturbance (3/12), dizziness (2/12), and impaired vision (1/12). GIECM were located frontal (6/12), temporal (3/12), parietal, fronto-parietal, and parieto-occipital (1/12 each). Microsurgical resection with acrylic-augmented cranioplasty was performed in all patients and 11/12 patients received dural repair with synthetic (7/11) or autologous (4/11) patch grafts. Surgical excision in two stages with primary removal of the extracranial meningioma component was undertaken in 2/12 patients, whereas preoperative embolization and postoperative radiotherapy were applied in 1/12 patient each. Results: In contrast to intradural meningiomas GIECM mainly affect male patients at a comparatively younger age. GIECM could be completely (9/12) or subtotally (3/12) resected. Surgical-associated complications included minor CSF leak (6/12), wound healing disturbance (3/12), venous engorgement, and haemorrhage (2/12 each), requiring reoperation in 3/12 cases. Histopathological examination revealed meningothelial (6/12), atypical (4/12), and transitional (1/12) GIECM. 10/12 patients exhibited excellent postoperative clinical outcome, 1/12 patient each deteriorated or died of pulmonary embolism. Conclusions: The operative management of GIECM is challenging, carries a substantial risk, and demands special strategies because of the large tumour size, anatomical involvement of scalp, calvaria, meninges, brain or vascular structures, and more frequent atypical histology. Although microsurgical resection with cranioplasty and mostly dural grafting usually results in a good clinical outcome, the potential complication rate is markedly higher when compared to smaller meningiomas without extracranial component. Preoperative embolization and staging of surgical resection are possible additional therapeutic options.

The extended diep flap: extending the possibilities for breast reconstruction with tissue from the lower abdomen

The classical DIEP-flap is considered state-of-the-art in microsurgical autologous breast reconstruction. Some patients may require additional volume to match the contralateral breast. This quality control study prospectively evaluates the feasibility and outcome of a surgical technique, which pursues the volumetric augmentation of the DIEP-flap by harvesting of additional subscarpal fat tissue cranial to the classical flap border.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.

Safety and efficacy of allogeneic cell therapy in infarcted rats transplanted with mismatched cardiosphere-derived cells

Cardiosphere-derived cells (CDCs) are an attractive cell type for tissue regeneration, and autologous CDCs are being tested clinically. However, autologous therapy necessitates patient-specific tissue harvesting and cell processing, with delays to therapy and possible variations in cell potency. The use of allogeneic CDCs, if safe and effective, would obviate such limitations. We compared syngeneic and allogeneic CDC transplantation in rats from immunologically-mismatched inbred strains.

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 × 10(6) 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.

Translation from research to applications

The article summarizes the collective views expressed at the fourth session of the workshop Tissue Engineering-the Next Generation, which was devoted to the translation of results of tissue engineering research into applications. Ernst Hunziker described the paradigm of a dual translational approach, and argued that tissue engineering should be guided by the dimensions and physiological setting of the bodily compartment to be repaired. Myron Spector discussed collagen-glycosaminoglycan (GAG) scaffolds for musculoskeletal tissue engineering. Jeanette Libera focused on the biological and clinical aspects of cartilage tissue engineering, and described a completely autologous procedure for engineering cartilage using the patient's own chondrocytes and blood serum. Arthur Gertzman reviewed the applications of allograft tissues in orthopedic surgery, and outlined the potential of allograft tissues as models for biological and medical studies. Savio Woo discussed a list of functional tissue engineering approaches designed to restore the biochemical and biomechanical properties of injured ligaments and tendons to be closer to that of the normal tissues. Specific examples of using biological scaffolds that have chemoattractants as well as growth factors with unique contact guidance properties to improve their healing process were shown. Anthony Ratcliffe discussed the translation of the results of research into products that are profitable and meet regulatory requirements. Michael Lysaght challenged the proposition that commercial and clinical failures of early tissue engineering products demonstrate a need for more focus on basic research. Arthur Coury described the evolution of tissue engineering products based on the example of Genzyme, and how various definitions of success and failure can affect perceptions and policies relative to the status and advancement of the field of tissue engineering.

Mechanical and radiological assessment of the influence of rhTGFbeta-3 on bone regeneration in a segmental defect in the ovine tibia: pilot study

Limitations in the use of autologous bone graft, which is the gold standard therapy in bone defect healing, drive the search for alternative treatments. In this study the influence of rhTGFbeta-3 on mechanical and radiological parameters of a healing bone defect in the sheep tibia was assessed. In the sheep, an 18-mm long osteoperiosteal defect in the tibia was treated by rhTGFbeta-3 seeded on a poly(L/DL-lactide) carrier (n = 4). In a second group (n = 4), the defect was treated by the carrier only, in a third group (n = 4) by autologous cancellous bone graft, and in a fourth group (n = 2) the defect remained blank. The healing process of the defect was assessed by weekly in vivo stiffness measurements and radiology as well as by quantitative computed tomographic assessment of bone mineral density (BMD) every 4 weeks. The duration of the experiment was 12 weeks under loading conditions. In the bone graft group, a marginally significant higher increase in stiffness was observed than in the PLA/rhTGFbeta-3 group (p = 0.06) and a significantly higher increase than in the PLA-only group (p = 0.03). The radiographic as well as the computed tomographic evaluation yielded significant differences between the groups (p = 0.03), indicating the bone graft treatment (bone/per area, 83%; BMD, 0.57 g/cm(3)) performing better than the PLA/rhTGFbeta-3 (38%; 0.23 g/cm(3)) and the PLA-only treatment (2.5%; 0.09 g/cm(3)), respectively. Regarding the mechanical and radiological parameters assessed in this study, we conclude that rhTGFbeta-3 has a promoting effect on bone regeneration. However, under the conditions of this study, this effect does not reach the potential of autologous cancellous bone graft transplantation.

A dedicated animal model for mechanical thrombectomy in acute stroke

BACKGROUND: Recent studies have focused on mechanical thrombectomy as a means to reduce the time required for revascularization and increase the revascularization rate in acute stroke. To date no systematic evaluation has been made of the different mechanical devices in this novel and fast-developing field of endovascular interventions. To facilitate such evaluations, we developed a specific in vivo model for mechanical thrombectomy that allows visualization of dislocation or fragmentation of the thrombus during angiographic manipulation. METHODS: Angiography and embolization with a preformed thrombus was performed in 8 swine. The thrombus was generated by mixing 25 IU bovine thrombin and 10 mL autologous blood. For visualization during angiography, 1 g barium sulfate was added. RESULTS: The preformed thrombus exhibited mechanical stability, reproducibility, and high radiographic absorption, providing excellent visibility during angiography. The setting allowed selective embolization of targeted vessels without thrombus fragmentation. Despite the application of barium sulfate no local or systemic reaction occurred. Histologic evaluation revealed no intimal damage caused by the thrombus or contrast agent washout. CONCLUSION: The model presented here allows selective and reliable thromboembolization of vessels that reproduce the anatomic and hemodynamic situation in acute cerebrovascular stroke. It permits visualization of the thrombus during angiography and intervention, providing unique insight into the behavior of both thrombus and device, which is potentially useful in the development and evaluation of mechanical clot retrieval in acute cerebrovascular stroke.

Expanded polytetrafluoroethylene graft for bypass surgery using the excimer laser-assisted nonocclusive anastomosis technique

OBJECT: Patients with complex craniocerebral pathophysiologies such as giant cerebral aneurysms, skull base tumors, and/or carotid artery occlusive disease are candidates for a revascularization procedure to augment or preserve cerebral blood flow. However, the brain is susceptible to ischemia, and therefore the excimer laser-assisted nonocclusive anastomosis (ELANA) technique has been developed to overcome temporary occlusion. Harvesting autologous vessels of reasonable quality, which is necessary for this technique, may at times be problematic or impossible due to the underlying systemic vascular disease. The use of artificial vessels is therefore an alternative graft for revascularization. Note, however, that it is unknown to what degree these grafts are subject to occlusion using the ELANA anastomosis technique. Therefore, the authors studied the ELANA technique in combination with an expanded polytetrafluoroethylene (ePTFE) graft. METHODS: The experimental surgeries involved bypassing the abdominal aorta in the rabbit. Ten rabbits were subjected to operations representing 20 ePTFE graft-ELANA end-to-side anastomoses. Intraoperative blood flow, followup angiograms, and long-term histological characteristics were assessed 75, 125, and 180 days postoperatively. Angiography results proved long-term patency of ePTFE grafts in all animals at all time points studied. Data from the histological analysis showed minimal intimal reaction at the anastomosis site up to 180 days postoperatively. Endothelialization of the ePTFE graft was progressive over time. CONCLUSIONS: The ELANA technique in combination with the ePTFE graft seems to have favorable attributes for end-to-side anastomoses and may be suitable for bypass procedures.

Improved neo-endothelialization of small diameter ePTFEgrafts with titanium coating

BACKGROUND: Patency of small synthetic bypass grafts is inferior compared to autologous grafts for revascularization procedures. Titanium coating of foreign surfaces has shown to decrease thrombogenicity, enhance biocompatibility and promote adhesion of endothelial cells. The aim of this study was to test the effect of titanium coating of small diameter ePTFE grafts on short term patency, neo-endothelialization and neointimal proliferation. METHODS: Bilateral carotid graft interposition was performed in 5 pigs with uncoated (n=5) and titanium-coated (n=5) ePTFE grafts (internal diameter=4 mm, length=5 cm), thus each pig served as its own control. At the end of the study (30 +/- 3 days), patency and stenosis severity was assessed by carotid angiography. Animals were sacrificed and grafts were excised for histology and scanning electron microscopy. Morphometry of histologic sections was carried out to determine neointimal proliferation and percentage of neo-endothelial coverage. RESULTS: Patency rate was 80% for uncoated and titanium-coated grafts. Quantitative angiography did not show any significant difference in lumen size between two groups. Morphometry revealed a significantly higher cellular coverage with CD31 positive endothelial cells for titanium-coated (84 +/- 19%) than uncoated grafts (48 +/- 26%, p<0.001). There was a non significant trend (p=0.112) towards increased neointimal proliferation in titanium-coated (94 +/- 61 micron2/micron) compared to uncoated grafts (60 +/- 57 micron2/micron). CONCLUSIONS: Patency rate in uncoated and titanium-coated ePTFE grafts is similar at one month. However, titanium coated grafts show a significant improvement in neo-endothelialization compared to uncoated grafts.

Evaluation of late results in breast reconstruction by latissimus dorsi flap and prosthesis implantation

BACKGROUND: Breast reconstruction by latissimus dorsi myocutaneous flap in combination with a prosthesis is a widely used, well-established procedure. Short- and medium-term evaluation after this procedure is well described in the literature, but there have been no evaluations of the late course (over 10 years) published until now. METHODS: In a retrospective study, 68 patients operated on by means of this technique at the authors' institution from 1981 to 1993 resulting in a minimal follow-up of 10 years were included. Patients were invited to an interrogation, clinical examination, and photographic documentation (n = 51). Incidence of late flap or prosthesis-related complications, number of and indications for corrective procedures, and the correlation of the patients' subjective judgment and objective results in the late course have been the main interest of the authors' survey. RESULTS: The authors found that 50 percent of the patients needed a late reoperation for change or removal of the prosthesis. Seven (10 percent) of 68 patients needed a definitive removal of the implant in the late course. Assessment of the photographic documentation of the late result by four nonprofessionals showed that the objective aesthetic results of a considerable number of the authors' reconstructions were not sufficient. CONCLUSION: The procedure combines two basic techniques of reconstructive surgery, the soft-tissue restoration by a pedicled flap as the autologous reconstructive component and the volume reconstruction by prosthesis. Therefore, these patients are subject to a cumulation of the basic morbidity of the two techniques. The authors conclude that the indication for this procedure should be restricted to patients not qualifying for "pure" reconstructive techniques.

Effects of local continuous release of brain derived neurotrophic factor (BDNF) on peripheral nerve regeneration in a rat model

The purpose of this study was to evaluate the effect of continuously released BDNF on peripheral nerve regeneration in a rat model. Initial in vitro evaluation of calcium alginate prolonged-release-capsules (PRC) proved a consistent release of BDNF for a minimum of 8 weeks. In vivo, a worst case scenario was created by surgical removal of a 20-mm section of the sciatic nerve of the rat. Twenty-four autologous fascia tubes were filled with calcium alginate spheres and sutured to the epineurium of both nerve ends. The animals were divided into 3 groups. In group 1, the fascial tube contained plain calcium alginate spheres. In groups 2 and 3, the fascial tube contained calcium alginate spheres with BDNF alone or BDNF stabilized with bovine serum albumin, respectively. The autocannibalization of the operated extremity was clinically assessed and documented in 12 additional rats. The regeneration was evaluated histologically at 4 weeks and 10 weeks in a blinded manner. The length of nerve fibers and the numbers of axons formed in the tube was measured. Over a 10-week period, axons have grown significantly faster in groups 2 and 3 with continuously released BDNF compared to the control. The rats treated with BDNF (groups 2 and 3) demonstrated significantly less autocannibalization than the control group (group 1). These results suggest that BDNF may not only stimulate faster peripheral nerve regeneration provided there is an ideal, biodegradable continuous delivery system but that it significantly reduces the neuropathic pain in the rat model.

Cartilage tissue engineering for degenerative joint disease

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.