Maurizio Calcagni

Morbidity and recurrence after completion lymph node dissection following sentinel lymph node biopsy in cutaneous malignant melanoma.

Objective:To assess the nature and rates of complications and recurrences after completion lymph node dissection (CLND) following positive sentinel lymph node biopsy (SLNB) in melanoma patients. Summary Background Data:In contrast to SLNB, CLND is associated with considerable morbidity. CLND delays nodal recurrence, thereby prolonging disease-free survival (DFS), but not overall melanoma-specific survival. Elaborate studies on morbidity and recurrence rates after CLND are scarce. Therefore, many controversies concerning extent and nature of CLND exist. Methods:We conducted a retrospective study on 100 melanoma patients, on whom we performed CLND between October 1999 and December 2005. The median observation period was 38.8 months. Results:We performed a total of 102 CLNDs, [46.1% axillary (47/102), 42.2% groin (43/102), 11.8% neck (12/102)]. Groin dissection (GD) and axillary dissection (AD) led to comparable morbidity (47.6% and 46.8%), but complications were more severe in GD, mandating additional surgery in 25.6% (11/43), versus 8.5% (4/47) in AD. Of the GD patients, 18.5% (8/43) were readmitted for complications compared with 10.4% (5/47) of AD patients. Only 8.3% (1/12) of ND patients suffered complications, mandating neither readmittance nor further surgery. During the median observation period, 65 (65%) of these patients showed DFS, and 35 (35%) exhibited recurrences after a median DFS of 12.5 months. Of the recurrences, 31.4% were nodal, 42.9% distant, and 25.7% local/in-transit. Of our AD patients, 28.3% suffered recurrences (13/46), as did 33.3% of the GD (14/42) and 66.7% of the ND patients (8/12). Conclusions:CLND is fraught with considerable morbidity. Local control of the dissected nodal basins was achieved with a modified radical approach in ADs (levels I + II only) and, to a lesser extent, GDs, but not in NDs. Clinical trials are necessary to establish guidelines on the extent of lymphatic dissection.

Tissue engineered bone grafts based on biomimetic nanocomposite PLGA/amorphous calcium phosphate scaffold and human adipose-derived stem cells.

For tissue engineering of critical size bone grafts, nanocomposites are getting more and more attractive due to their controllable physical and biological properties. We report in vitro and in vivo behaviour of an electrospun nanocomposite based on poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) seeded with human adipose-derived stem cells (ASC) compared to PLGA. Major findings were that cell attachment, three-dimensional ingrowth and proliferation were very good on both materials. Cell morphology changed from a spindle-shaped fibroblast-like form to a more roundish type when ASC were seeded on PLGA, while they retained their morphology on PLGA/a-CaP. Moreover, we found ASC differentiation to a phenotype committed towards osteogenesis when a-CaP nanoparticles were suspended in normal culture medium without any osteogenic supplements, which renders a-CaP nanoparticles an interesting osteoinductive component for the synthesis of other nanocomposites than PLGA/a-CaP. Finally, electrospun PLGA/a-CaP scaffold architecture is suitable for a rapid and homogenous vascularisation confirmed by a complete penetration by avian vessels from the chick chorioallantoic membrane (CAM) within one week.

A new model for studying the revascularization of skin grafts in vivo: the role of angiogenesis.

Background: Models of skin graft revascularization are based mostly on histologic evaluations but lack the possibility of analyzing the vascular biology in vivo. The aim of the present study was therefore to develop an animal model that allows continuous monitoring of the microcirculation during skin graft healing. Methods: Skin and subcutaneous tissue were removed from the back of dorsal skinfold chamber preparations in mice, leaving one layer of striated muscle and subcutaneous tissue as a wound bed (n = 5). A corresponding full-thickness skin graft was harvested from the groin and sutured into the defect in the back of the chamber. To study graft healing, repetitive intravital microscopy was performed during the first 10 days after engraftment. Results: Capillary widening in the wound bed appeared at day 1 after grafting and increased until day 4. Capillary buds and sprouts first appeared at day 2. Blood filling of autochthonous graft capillaries occurred at day 3, resulting in almost complete restoration of the original skin microcirculation on day 5. This was achieved by interconnections between the microvasculature of the wound bed and the skin graft through a temporary angiogenic response. In principle, angiogenic blood vessel growth originated in the wound bed and was directed toward the graft. Conclusions: This new model allows for repetitive analysis of the microcirculation during skin graft healing. It provides ideal in vivo conditions to further delineate the exact mechanisms of blood vessel interconnection during the complex process of angiogenesis, and may also allow study of the vascularization of tissue-engineered skin substitutes.

Suitability of Thiel embalmed tendons for biomechanical investigation.

The standard post-mortem storage method for biomechanical testing is freezing. Freezing minimally alters the biomechanical characteristics of tendons but only suspends the process of decay. Chemical fixation arrests decay and overcomes risk of infection, but alters the biomechanical properties of tendons. On the other hand, Thiel preservation has been reported to maintain soft tissue consistency similar to that of living tissue. The current study investigates the effects of Thiel embalming on human digitorum profundus tendons (FDP) from fresh-frozen and Thiel embalmed cadavers. Cross-sectional area was measured at pre-load, samples were preconditioned and then ramped at a constant strain-rate to failure. Thiel preserved tendons had statistically lower failure stress with median of 38MPa compared to fresh frozen samples with median of 60MPa (p-value=0.048) and trended to a decreased tangential modulus. To overcome limited donor number and masking factors of age, gender, and time embalmed, we also performed experiments in rat tail tendon fascicle. Similar quasi-static ramp to failure tests were performed with control and Thiel treated sample pairs. Similar differences were observed to those found as in human FDP, however these trends were statistically significant. In both tendons, Thiel preserved samples demonstrated altered failure characteristics, indicating a different collagen fiber/collagen network failure mechanism most likely due to partial denaturing by boric acid in Thiel solution. In conclusion, Thiel embalmed tendons did not faithfully represent the biomechanical characteristics of fresh frozen tendons.

Yield and proliferation rate of adipose-derived stromal cells as a function of age, body mass index and harvest site—increasing the yield by use of adherent and supernatant fractions?

BACKGROUND AIMS Adipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cells ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed. METHODS The surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined. RESULTS Both adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site. CONCLUSIONS The floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate.

Primary Flexor Tendon Repair in Zones 1 and 2: Early Passive Mobilization Versus Controlled Active Motion

PURPOSE To compare early passive mobilization (EPM) with controlled active motion (CAM) after flexor tendon surgery in zones 1 and 2. METHODS We performed a retrospective analysis of collected data of all patients receiving primary flexor tendon repair in zones 1 and 2 from 2006 to 2011, during which time 228 patients were treated, and 191 patients with 231 injured digits were eligible for study. Exclusion criteria were replantation, finger revascularization, age younger than 16 years, rehabilitation by means other than EPM or CAM, and missing information regarding postoperative rehabilitation. This left 132 patients with 159 injured fingers for analysis. The primary endpoint was the comparison of total active motion (TAM) values 4 and 12 weeks after surgery between the EPM and the CAM protocols. The analysis of TAM measurements under the rehabilitation protocols was conducted using t-tests and further linear modeling. We defined rupture rate and the assessment of adhesion/infection as secondary endpoints. RESULTS There was a statistically significant difference between the TAM values of the EPM and the CAM protocols 4 weeks after surgery. At 12 weeks, however, there was no significant difference between the 2 protocols. Older age and injuries with finger fractures were associated with lower TAM values. Rupture rates were 5% (CAM) and 7% (EPM), which were not statistically different. CONCLUSIONS This study showed a favorable effect of CAM protocol on TAM 4 weeks after surgery. The percent rupture rate was slightly lower in the patients with CAM than in the patients with EPM regime. Further studies are required to confirm our results and to investigate whether faster recovery of TAM is associated with shorter time out of work. TYPE OF STUDY/LEVEL OF EVIDENCE Therapeutic III.

Human adipose-derived mesenchymal stromal cells may promote breast cancer progression and metastatic spread

Background: Stem cell–enriched fat grafting has been proposed as a potential therapy for reconstructive, restorative, or enhancement-related procedures of the breast. Its role in postoncologic breast reconstruction is still emerging, with concerns about safety. The authors investigated the dose-dependent interaction between human adipose-derived mesenchymal stromal cells (AD-MSCs) and human breast cancer cell (BCC) lines [MDA-MB-231 (MDA) and MCF-7 (MCF)] focusing on tumor microenvironment, tumor growth, and metastatic spread. Methods: Adipose-derived mesenchymal stromal cell influence on viability and factor expression [regulated on activation, normal T cell expressed and secreted (RANTES), tumor necrosis factor-&agr;, and eotaxin) of breast cancer cells was studied in vitro using direct and indirect co-culture systems. Groups were formed according to adipose-derived mesenchymal stromal cell–to–cancer cell number ratio [MDA/MCF only, AD-MSClow/(MDA/MCF), and AD-MSChigh/(MDA/MCF)]. A humanized orthotopic murine cancer model was used to evaluate breast cancer progression and metastasis (n = 10/group). Cells were injected into the mammary pad in different ratios and animals were monitored over 42 days. Microdialysis was performed to analyze RANTES levels in the tumor microenvironment (days 21 and 42). Primary and metastatic tumors were weighed and analyzed for oncogene, growth factor, and metastatic marker expression. Results: MDA cell viability increased from 45.5 percent to 95.5 percent in presence of adipose-derived mesenchymal stromal cells in vitro. In vivo, animals with AD-MSChigh showed increased mean tumor weight (MDA, p < 0.01; MCF versus controls, p < 0.05) and metastatic occurrence (40 percent in MDA; 30 percent in MCF versus 0 percent in controls). Cytokine analysis revealed switching of MCF tumor phenotype to a more malignant type in the presence of adipose-derived mesenchymal stromal cells. Conclusion: Human adipose-derived mesenchymal stromal cells may promote progression and metastatic spread in breast cancer through a switch to a more malignant phenotype with worse prognosis.

In vivo visualization of the origination of skin graft vasculature in a wild-type/GFP crossover model

INTRODUCTION Skin substitutes are increasingly produced in tissue engineering, but still the understanding of the physiological skin revascularization process is lacking. To study in vivo conditions we recently introduced a mouse model, in which we already characterized the angiogenic changes within the wound bed and the skin graft. The aim of this study was to identify the origination of the vasculature during skin graft revascularization in vivo and to track vessel development over time. METHODS We created a crossover wild-type/GFP skin transplantation model, in which each animal carried the graft from the other strain. The preparation of the modified dorsal skin fold chamber including cross-over skin grafting was performed in male C57BL/6J wild-type mice (n=5) and C57BL/6-Tg(ACTB-EGFP)1O sb/J mice (n=5). Intravital microscopy in 12 areas of wild-type and GFP skin grafts was performed daily over a time period of 10 days. RESULTS Graft reperfusion started after 48-72 h. After reperfusion GFP-positive structures from the wound bed were visible in the graft capillaries with the highest density in the center of the graft. Overall, we observed a replacement of existing graft capillaries with vessels from the wound bed in 68% of the vessels. Of note, vessel replacement occurred in almost 100% of graft vessels in the periphery. Additionally, vessels within the graft showed a temporary angiogenic response between days 3-8, which originated predominantly from the autochthonous graft vasculature, but also contained already grown-in vessels from the wound bed. CONCLUSIONS These in vivo data indicate an early in-growth of angiogenic bed vessels into the existing vascular channels of the graft and subsequent centripetal replacement. Additionally we observed a temporary angiogenic response of the autochthonous capillaries of the skin graft with contribution from bed vessels. These findings further support the theory that sprouting angiogenesis from the wound bed in combination with the replacement of existing graft vessels are the key factors in skin graft taking. Thus, manufacturing of skin substitutes should be aimed at providing pre-formed vascular channels within the construct to improve vascularization.

Temporary angiogenic transformation of the skin graft vasculature after reperfusion.

Background: In the era of tissue engineering, the physiologic process of skin graft revascularization remains unclear, preventing the successful development of skin substitutes. Therefore, the authors developed a new in vivo model with which to visualize the process of engraftment and its microvascular architecture. The aim of this study was to specifically investigate the vascular transformations within the skin graft to gain applicable knowledge on how vascular processes during engraftment occur. Methods: Microsurgical preparation of the modified dorsal skinfold chamber including autologous skin grafting was performed in male C57BL/6J mice (n = 10). In addition, immunohistochemistry of angiogenic factors, endothelial cells, and pericytes, and corrosion casting were performed to further characterize the specific mechanisms. Results: The graft exhibited capillary widening starting at day 3, resulting in the temporary formation of spherical protrusions at the graft capillary divisions starting in the center of the graft 24 to 48 hours after revascularization. Confocal microscopy showed the simultaneous expression of CD31 and desmin. Corrosion casting and evaluation by light microscopy and scanning electron microscopy showed the three-dimensional formation of capillaries in the wound bed that connected to the preexisting capillary loops of the skin graft. Conclusions: The authors were able to show for the first time a temporary angiogenic response within the capillaries of the skin graft. This most likely represents a reaction to reperfusion allowing the supply of proangiogenic factors to the hypoxic skin graft. The detection of an angiogenic response within the graft capillaries is for the first time made possible in the newly developed model and is therefore completely novel.

Cellular response of healing tissue to DegraPol tube implantation in rabbit Achilles tendon rupture repair: an in vivo histomorphometric study

In tendon rupture repair, improvements such as higher primary repair strength, anti‐adhesion and accelerated healing are needed. We developed a potential carrier system of an electrospun DegraPol® tube, which was tightly implanted around a transected and conventionally sutured rabbit Achilles tendon. Histomorphometric analysis of the tendon tissue 12 weeks postoperation showed that the tenocyte density, tenocyte morphology and number of inflammation zones were statistically equivalent, whether or not DegraPol tube was implanted; only the collagen fibres were slightly less parallelly orientated in the tube‐treated case. Comparison of rabbits that were operated on both hind legs with ones that were operated on only one hind leg showed that there were significantly more inflammation zones in the two‐leg cases compared to the one‐leg cases, while the implantation of a DegraPol tube had no such adverse effects. These findings are a prerequisite for using DegraPol tube as a carrier system for growth factors, cytokines or stem cells in order to accelerate the healing process of tendon tissue. Copyright