Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Presentation, microsurgical therapy, and clinical outcomes in three cases of expanding melanonychia of the nail unit in children

The management of expanding melanonychia in childhood is controversial. Here, we present three cases and discuss their operating indications and reconstruction. Between January 1, 1995 and December 31, 2007, one boy and two girls, were operated for expanding melanonychia, involving the thumb, index finger or the middle finger. They were 2, 4, and 7 years at the time of surgery. A complete resection of the nail plate was performed followed by a direct finger reconstruction using a free short-pedicle vascularized nail flap of the toe. Histology showed a junctional nevus in all cases. The follow-ups were after 2, 3, and 5 years and without any complications or recurrence. Regarding reconstruction, the mean Foucher and Leclère score were, respectively, 17 and 16 points. It is concluded that for expanding melanonychia, in case of doubt, an examination of the entire lesion is necessary. Reconstruction of the nail unit after wide excision with nail plate ablation can be performed using microsurgery as discussed below. However, new guidelines on shave biopsy can make this microsurgical procedure obsolete.

1.9 µm diode laser assisted vascular microanastomoses: Experience in 40 clinical procedures

Since the initial work of Jacobson and Suarez in 1960, microsurgery has evolved greatly. In 2009, we reported our clinical experience with 1.9 µm diode laser-assisted vascular microanastomoses (LAMA) for free flap reconstruction. In this report, the ongoing study is now expanded to include 11 additional procedures which were analyzed prospectively with a focus on the duration of the LAMA technique.

[Blood flow assessment with magnetic resonance imaging after 1.9 μm diode laser assisted arterial micronastomoses]

The most important factor for successful free-flap transfer and replantations is a well-executed anastomosis. The aim of this study is to assess blood flow after laser assisted arterial microanastomosis (LAMA) using a 1.9 μm diode laser.

[Historical review and future orientations of the conventional vascular microanastomoses]

Microvascular surgery has become an important method for reconstructing surgical defects due to trauma, tumors or after burn. The most important factor for successful free flap transfer is a well-executed anastomosis. The time needed to perform the anastomosis and the failure rate are not negligible despite the high level of operator's experience. During the history, many alternatives were tried to help the microsurgeon and to reduce the complications. A Medline literature search was performed to find articles dealing with non-suture methods of microvascular anastomosis. Many historical books were also included. The non-suture techniques can be divided into four groups based on the used mechanism of sutures: double intubation including tubes and stents, intubation-eversion including simple rings, double eversion including staples and double rings, and wall adjustement with adhesives or laser. All these techniques were able to produce a faster and easier microvascular anastomosis. Nevertheless, disadvantages of the suturless techniques include toxicity, high cost, leakage or aneurysm formation. More refinement is needed before their widespread adoption. Thus, laser-assisted microvascular anastomosis using 1,9 μm diode laser appeared to be a safe and reliable help for the microsurgeon and may be further developed in the near future.

Patient-centered outcomes after subepithelial connective tissue grafts and coronally advanced flaps

The use of a coronally advanced flap (CAF) and connective tissue graft (CTG) is a well-established procedure to cover single and multiple recessions and improve soft tissue esthetics. However, until now, there are still limited data evaluating patient morbidity, the fear of imminent tooth loss, and modification of sensitivity in surgical areas. The aim of the present study was to evaluate the patient-centered outcomes associated with CAF + CTG.

Treatment of soft tissue recessions at titanium implants using a resorbable collagen matrix: a pilot study

OBJECTIVES: To histologically assess the effectiveness of a porcine-derived collagen matrix (CM) and a subepithelial connective tissue graft (CTG) for the coverage of single mucosal recessions at osseointegrated dental implants. MATERIALS AND METHODS: Chronic-type mucosal Miller Class I-like recessions (mean clinical defect height: 0.67 ± 0.33-1.16 ± 0.19 mm) were established at the buccal aspect of titanium implants with platform switch in six beagle dogs. The defects were randomly allocated to either (1) coronally advanced flap surgery (CAF) + CM, (2) CAF + CTG or (3) CAF alone. At 12 weeks, histomorphometrical measurements were made (e.g.) between the implant shoulder (IS) and the mucosal margin (PM) and IS and the outer contour of the adjacent soft tissue (mucosal thickness [MT]). RESULTS: All treatment procedures investigated were associated with an almost complete soft tissue coverage of the defect area (i.e. coronal positioning of PM relative to IS). Mean IS-PM and MT values tended to be increased in both CAF + CM (1.04 ± 0.74 mm/0.71 ± 0.55 mm) and CAF + CTG (0.88 ± 1.23 mm/0.62 ± 0.66 mm) groups when compared with CAF (0.16 ± 0.28 mm/0.34 ± 0.23 mm) alone. These differences, however, did not reach statistical significance. CONCLUSIONS: Within the limits of this pilot study, it was concluded that all treatment procedures investigated were effective in covering soft tissue recessions at titanium implants.

A phase IIa randomized controlled pilot study evaluating the safety and clinical outcomes following the use of rhGDF-5/β-TCP in regenerative periodontal therapy

To present the safety profile, the early healing phase and the clinical outcomes at 24 weeks following treatment of human intrabony defects with open flap debridement (OFD) alone or with OFD and rhGDF-5 adsorbed onto a particulate β-tricalcium phosphate (β-TCP) carrier. Twenty chronic periodontitis patients, each with at least one tooth exhibiting a probing depth ≥6 mm and an associated intrabony defect ≥4 mm entered the study. Ten subjects (one defect/patient) were randomized to receive OFD alone (control) and ten subjects OFD combined with rhGDF-5/β-TCP. Blood samples were collected at screening, and at weeks 2 and 24 to evaluate routine hematology and clinical chemistry, rhGDF-5 plasma levels, and antirhGDF-5 antibody formation. Plaque and gingival indices, bleeding on probing, probing depth, clinical attachment level, and radiographs were recorded pre- and 24 weeks postsurgery. Comparable safety profiles were found in the two treatment groups. Neither antirhGDF-5 antibody formation nor relevant rhGDF-5 plasma levels were detected in any patient. At 6 months, treatment with OFD + rhGDF-5/β-TCP resulted in higher but statistically not significant PD reduction (3.7 ± 1.2 vs. 3.1 ± 1.8 mm; p = 0.26) and CAL gain (3.2 ± 1.7 vs. 1.7 ± 2.2 mm; p = 0.14) compared to OFD alone. In the tested concentration, the use of rhGDF-5/β-TCP appeared to be safe and the material possesses a sound biological rationale. Thus, further adequately powered, randomized controlled clinical trials are warranted to confirm the clinical relevance of this new approach in regenerative periodontal therapy. rhGDF-5/β-TCP may represent a promising new techology in regenerative periodontal therapy.

Comparison of implant stability by means of resonance frequency analysis for flapless and conventionally inserted implants

Background: Resonance frequency analysis (RFA) is a noninvasive technique for the quantitative assessment of implant stability. Information on the implant stability quotient (ISQ) of transmucosally inserted implants is limited. Purpose: The aim of this investigation was to compare the ISQ of conventionally inserted implants by raising a muco-periostal flap with implants inserted using a flapless procedure. Materials and Methods: Forty elderly patients with complete edentulous maxilla were consecutively admitted for treatment with implant-supported prostheses. A computer tomography was obtained for the computer-assisted implant planning. One hundred ten implants were placed conventionally in 23 patients (flap-group) and 85 implants in 17 patients by means of the flapless method (flapless-group) using a stereolithographic template. RFA measurements were performed after implant placement (baseline) and after a healing time of 12 weeks (reentry). Results: All implants exhibited clinically and radiographically successful osseointegration. Bone level did not change significantly neither for genders nor type of surgical protocol. Mean ISQ values of the flapless-group were significantly higher at baseline (p < .001) and at reentry (p < .001) compared with the flap-group. The ISQ values were significantly lower at reentry compared with baseline for the flap-group (p = .028) but not for the flapless-group. This group showed a moderate, but insignificant increase. RFA measurements of males resulted in ISQ values that were thoroughly higher as compared with females at both time-points in both groups. Correlation between RFA and bone level was not found. Conclusions: The flapless procedure showed favorable conditions with regard to implant stability and crestal bone level. Some changes of the ISQ values that represent primary (mechanical) and secondary (bone remodeling) implant stability were observed in slight favor of the flapless method and male patients. In properly planned and well-selected cases, the minimal invasive transmucosal technique using a drill-guide is a safe procedure.

Outcome and patterns of failure after postoperative intensity modulated radiotherapy for locally advanced or high-risk oral cavity squamous cell carcinoma

Background To determine the outcome and patterns of failure in oral cavity cancer (OCC) patients after postoperative intensity modulated radiotherapy (IMRT) with concomitant systemic therapy. Methods All patients with locally advanced (AJCC stage III/IV) or high-risk OCC (AJCC stage II) who underwent postoperative IMRT at our institution between December 2006 and July 2010 were retrospectively analyzed. The primary endpoint was locoregional recurrence-free survival (LRRFS). Secondary endpoints included distant metastasis-free survival (DMFS), overall survival (OS), acute and late toxicities. Results Overall 53 patients were analyzed. Twenty-three patients (43%) underwent concomitant chemotherapy with cisplatin, two patients with carboplatin (4%) and four patients were treated with the monoclonal antibody cetuximab (8%). At a median follow-up of 2.3 (range, 1.1–4.6) years the 3-year LRRFS, DMFS and OS estimates were 79%, 90%, and 73% respectively. Twelve patients experienced a locoregional recurrence. Eight patients, 5 of which had both a flap reconstruction and extracapsular extension (ECE), showed an unusual multifocal pattern of recurrence. Ten locoregional recurrences occurred marginally or outside of the high-risk target volumes. Acute toxicity grades of 2 (27%) and 3 (66%) and late toxicity grades of 2 (34%) and 3 (11%) were observed. Conclusion LRRFS after postoperative IMRT is satisfying and toxicity is acceptable. The majority of locoregional recurrences occurred marginally or outside of the high-risk target volumes. Improvement of high-risk target volume definition especially in patients with flap reconstruction and ECE might transfer into better locoregional control.

Intravascular ultrasound-guided creation of re-entry sites to improve intermittent claudication in patients with aortic dissection

PURPOSE: To report percutaneous fenestration of aortic dissection flaps to relieve distal ischemia using a novel intravascular ultrasound (IVUS)-guided fenestration device. CASE REPORTS: Two men (47 and 62 years of age) with aortic dissection and intermittent claudication had percutaneous ultrasound-guided fenestration performed under local anesthesia. Using an ipsilateral transfemoral approach, the intimal flap was punctured under real-time IVUS guidance using a needle-catheter combination through which a guidewire was placed across the dissection flap into the false lumen. The fenestration was achieved using balloon catheters of increasing diameter introduced over the guidewire. Stenting of the re-entry was performed in 1 patient to equalize pressure across the dissection membrane in both lumens. The procedures were performed successfully and without complications. In both patients, ankle-brachial indexes improved from 0.76 to 1.07 and from 0.8 to 1.1, respectively. Both patients were without claudication at the 3- and 6-month follow-up examination. CONCLUSION: Percutaneous intravascular ultrasound-guided fenestration and stenting at the level of the iliac artery in aortic dissection patients with claudication is a technically feasible and safe procedure and relieves symptoms.

Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: implantation method and short-term results

OBJECTIVE: To compare four different implantation modalities for the repair of superficial osteochondral defects in a caprine model using autologous, scaffold-free, engineered cartilage constructs, and to describe the short-term outcome of successfully implanted constructs. METHODS: Scaffold-free, autologous cartilage constructs were implanted within superficial osteochondral defects created in the stifle joints of nine adult goats. The implants were distributed between four 6-mm-diameter superficial osteochondral defects created in the trochlea femoris and secured in the defect using a covering periosteal flap (PF) alone or in combination with adhesives (platelet-rich plasma (PRP) or fibrin), or using PRP alone. Eight weeks after implantation surgery, the animals were killed. The defect sites were excised and subjected to macroscopic and histopathologic analyses. RESULTS: At 8 weeks, implants that had been held in place exclusively with a PF were well integrated both laterally and basally. The repair tissue manifested an architecture similar to that of hyaline articular cartilage. However, most of the implants that had been glued in place in the absence of a PF were lost during the initial 4-week phase of restricted joint movement. The use of human fibrin glue (FG) led to massive cell infiltration of the subchondral bone. CONCLUSIONS: The implantation of autologous, scaffold-free, engineered cartilage constructs might best be performed beneath a PF without the use of tissue adhesives. Successfully implanted constructs showed hyaline-like characteristics in adult goats within 2 months. Long-term animal studies and pilot clinical trials are now needed to evaluate the efficacy of this treatment strategy.

Significance of primary stability for osseointegration of dental implants

AIM: To investigate the significance of the initial stability of dental implants for the establishment of osseointegration in an experimental capsule model for bone augmentation. MATERIAL AND METHODS: Sixteen male rats were used in the study. In each rat, muscle-periosteal flaps were elevated on the lateral aspect of the mandibular ramus on both sides, resulting in exposure of the bone surface. Small perforations were then produced in the ramus. A rigid, hemispherical Teflon capsule with a diameter of 6 mm and a height of 4 mm and with a hole in its middle portion, prepared to fit the circumference of an ITI HC titanium implant of 2.8 mm in diameter, was fixed to the ramus using 4 mini-screws. On one side of the jaw, the implant was placed through the hole in such a way that its apex did not make contact with the mandibular ramus (test). This placement of the implant did not ensure primary stability. On the other side of the jaw, a similar implant was placed through the hole of the capsule in such a way that contact was made between the implant and the surface of the ramus (control). This provided primary stability of the implant. After placement of the implants, the soft tissues were repositioned over the capsules and sutured. After 1, 3, 6 and 9 months, four animals were sacrificed and subjected to histometric analysis. RESULTS: The mean height of direct bone-to-implant contact of implants with primary stability was 38.8%, 52.9%, 64.6% and 81.3% of the implant length at 1, 3, 6 and 9 months, respectively. Of the bone adjacent to the implant surface, 28.1%, 28.9%, 52.6% and 69.6%, respectively, consisted of mineralized bone. At the test implants, no bone-to-implant contact was observed at any observation time or in any of these non-stabilized specimens. CONCLUSION: The findings of the present study indicate that primary implant stability is a prerequisite for successful osseointegration, and that implant instability results in fibrous encapsulation, thus confirming previously made clinical observations.

Ridge augmentation by applying bioresorbable membranes and deproteinized bovine bone mineral: a report of twelve consecutive cases

OBJECTIVE: Lateral ridge augmentations are traditionally performed using autogenous bone grafts to support membranes for guided bone regeneration (GBR). The bone-harvesting procedure, however, is accompanied by considerable patient morbidity. AIM: The aim of the present study was to test whether or not resorbable membranes and bone substitutes will lead to successful horizontal ridge augmentation allowing implant installation under standard conditions. MATERIAL AND METHODS: Twelve patients in need of implant therapy participated in this study. They revealed bone deficits in the areas intended for implant placement. Soft tissue flaps were carefully raised and blocks or particles of deproteinized bovine bone mineral (DBBM) (Bio-Oss) were placed in the defect area. A collagenous membrane (Bio-Gide) was applied to cover the DBBM and was fixed to the surrounding bone using poly-lactic acid pins. The flaps were sutured to allow for healing by primary intention. RESULTS: All sites in the 12 patients healed uneventfully. No flap dehiscences and no exposures of membranes were observed. Nine to 10 months following augmentation surgery, flaps were raised in order to visualize the outcomes of the augmentation. An integration of the DBBM particles into the newly formed bone was consistently observed. Merely on the surface of the new bone, some pieces of the grafting material were only partly integrated into bone. However, these were not encapsulated by connective tissue but rather anchored into the newly regenerated bone. In all of the cases, but one, the bone volume following regeneration was adequate to place implants in a prosthetically ideal position and according to the standard protocol with complete bone coverage of the surface intended for osseointegration. Before the regenerative procedure, the average crestal bone width was 3.2 mm and to 6.9 mm at the time of implant placement. This difference was statistically significant (P<0.05, Wilcoxon's matched pairs signed-rank test). CONCLUSION: After a healing period of 9-10 months, the combination of DBBM and a collagen membrane is an effective treatment option for horizontal bone augmentation before implant placement.

The influence of local and systemic preconditioning on oxygenation, metabolism and survival in critically ischaemic skin flaps in pigs

Stress proteins represent a group of highly conserved intracellular proteins that provide adaptation against cellular stress. The present study aims to elucidate the stress protein-mediated effects of local hyperthermia and systemic administration of monophosphoryl lipid A (MPL) on oxygenation, metabolism and survival in bilateral porcine random pattern buttock flaps. Preconditioning was achieved 24h prior to surgery by applying a heating blanket on the operative site (n = 5), by intravenous administration of MPL at a dosage of 35 microg/kg body weight (n = 5) or by combining the two (n = 5). The flaps were monitored with laser Doppler flowmetry, polarographic microprobes and microdialysis until 5h postoperatively. Semiquantitative immunohistochemistry was performed for heat shock protein 70 (HSP70), heat shock protein 32 (also termed haem oxygenase-1, HO-1), and inducible nitrc oxide synthase (iNOS). The administration of MPL increased the impaired microcirculatory blood flow in the proximal part of the flap and partial oxygen tension in the the distal part by approximately 100% each (both P<0.05), whereas both variables remained virtually unaffected by local heat preconditioning. Lactate/pyruvate (L/P) ratio and glycerol concentration (representing cell membrane disintegration) in the distal part of the flap gradually increased to values of approximately 500 mmol/l and approximately 350 micromol/l, respectively (both P<0.01), which was substantially attenuated by heat application (P<0.01 for L/P ratio and P<0.05 for glycerol) and combined preconditioning (P<0.01 for both variables), whereas the effect of MPL was less marked (not significant). Flap survival was increased from 56% (untreated animals) to 65% after MPL (not significant), 71% after heat application (P<0.05) and 78% after both methods of preconditioning (P<0.01). iNOS and HO-1 were upregulated after each method of preconditioning (P<0.05), whereas augmented HSP70 staining was only observed after heat application (P<0.05). We conclude that local hyperthermia is more effective in preventing flap necrosis than systemic MPL administration because of enhancing the cellular tolerance to hypoxic stress, which is possibly mediated by HSP70, whereas some benefit may be obtained with MPL due to iNOS and HO-1-mediated improvement in tissue oxygenation.