International Journal of Cardiology 182 (2015) 466–468
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Novel application of mesenchymal stem cells from mammary artery Natalia Malara a,b, Giuseppe Musolino c, Valentina Trunzo a, Arturo Mario Minniti a, Lucia Cristodoro c, Massimo Polistina c, Attilio Renzulli c, Vincenzo Mollace a,⁎ a b c
Institute of Research for Food Safety and Health (IRC-FSH), Department of Health Science University “Magna Graecia” of Catanzaro, Italy Bionem Laboratories, Department of Experimental and Clinical Medicine, Salvatore Venuta Campus, University “Magna Graecia” of Catanzaro Italy Cardiac Surgery Unit, Department of Medical and Surgical Sciences, Salvatore Venuta Campus, University “Magna Graecia” of Catanzaro Italy
a r t i c l e
i n f o
Article history: Received 30 December 2014 Accepted 2 January 2015 Available online 6 January 2015 Keywords: Autologous mesenchymal stem cells Mammary artery Mediastinitis
The incidence of 1 to 4% of mediastinitis has been reported to occur as a consequence of cardio-thoracic surgery procedures. This complication continues to have a dramatic impact on patient outcomes reporting hospital mortality between 10 and 20% [1]. Recently, vacuum therapy and new techniques as wound dressing with plasma rich of platelets and wound reconstruction with autologous or heterologous tissue [2] have improved clinical results; nevertheless morbidity remains consistent and mortality still occurs in patients with co-morbidities following prolonged hospital stay. Thus, not-conventional approaches have been proposed to get better results in regenerative medicine applied to secondary mediastinitis. Here we report on recurrent sternotomy infection refractory to conventional treatments, successfully approached with an original protocol based on the use of autologous MSC derived from fragments of left internal mammary artery (LIMA) not utilized during the first CABG operation. A 83 year old, man, suffering from type 2 diabetes mellitus treated with insulin therapy, hypochromic anemia, hypertension, peripheral artery occlusive disease, three-vessel coronary artery disease, underwent on pump CABGx3 through median sternotomy. LIMA was harvested and implanted on the left anterior descending artery. Sequential venous graft was performed on posterior descending and obtuse marginal braches. Excess of the length of mammary artery was sent to the Human Cellular Biology Laboratory IRC-FSH1 to isolate and culture endothelial cells for further characterization. Moreover, in order to ⁎ Corresponding author at: IRC-FSH, University “Magna Graecia” of Catanzaro, Campus Universitario Germaneto, 88100 Catanzaro, Italy. E-mail address: [email protected] (V. Mollace). 1 Interregional Research Center for Food Safety and Health.
http://dx.doi.org/10.1016/j.ijcard.2015.01.007 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
reduce the risk of post-operative infection sterna wound was approximated over 5 ml of autologous platelet gel. Patient informed consent was previously obtained as part of “SCENIC2” project approved by ethical committee of “Mater Domini”. Biopsied vessels were immediately immersed in standard medium with complete F-12K growth medium, 0.1 mg/ml heparin, 0.03 mg/ml endothelial cell growth supplement (ECGS), fetal calf serum 10%, gentamicin 3%, amphotericin D 3% and rinsed with phosphate-buffered saline. The vessel fragment was placed in a culture dish (Fig. 1), the adipose tissue was removed, the vessel was de-endothelialized cutting lengthwise. The remaining layer was cut into 1.5 mm pieces. Each piece was placed in a 96-well culture plate adding 50 μL of fresh F-12K complete growth medium, supplemented with 5 μL (1:10) of ECGS. Successively cells were incubated at 37 °C and 5% CO2. Cytometric evaluation allowed cell surface characterization of adult MSC. CD44positive (45%), CD45negative (72%) and fibronectinpositive (53%) expression confirmed the mesenchymal profile. Post-operative course was initially uneventfully and patient was discharged home on the tenth postoperative day. Six days following hospital discharge, patient was readmitted with fever leucocitosis, sternal instability and purulent discharge from the sternal wound. Surgical debridement was performed, sterna wires were removed, and VAC therapy was started. Bacteriologic analyses showed an infection supported by Staphylococcus epidermidis and Staphylococcus hominis. VAC therapy and i.v. antibiotic infusion were continued for six weeks. Despite of negative tissue culture results, the sternal wound appeared still necrotic with few granulation tissue. At this point we performed wound closure over subcutaneous drainage. Nevertheless, after fourteen days we observed complete wound dehiscence (Fig. 2A). We were faced with a patient in advanced age, psychologically depressed. We discussed with him about the possibility to perform an alternative therapy to improve wound healing avoiding further procedures under general anesthesia. We proposed to him to close the wound under local anesthesia, and to implant autologous MSCs isolated in order to improve tissue replacement and vascularization. Therefore, wound was stitched under local anesthesia and skin was re-approximated using 4 full thickness treads of mersilene 5/0. Collected MSCs were injected just above the periosteum with 22G needle. Three injections for each sterna side were performed. We monitored the injection points of MSCs with ultrasonography technique through a 9 MHz transducer. MSCs at injection point appeared as hyper reflective nodule areas characterized by regular shape and 2
Standardization of Circulating ENdothelIal Cells evaluation.
N. Malara et al. / International Journal of Cardiology 182 (2015) 466–468
467
Fig. 1. (A) Excess of the length of mammary artery fragment immersed in standard medium. (B) In vitro expansion human primary endothelial cells. (C) In blue the nuclear staining with DAPI. (D) Immunofluorescence in red showed the surface positivity for CD146 expression, differentiated endothelial antigen. In vitro MSCs expanded (E) and stained with DAPI (F) to evidence nuclei. Cytometric evaluation of MSC phenotype. Dot plots showed MSCs in side scattering (SSC) and forward scattering (FSC), their negativity for CD45 and positivity expression for CD44 and fibronectin antigens.
maximum diameter of 0.88 cm (Fig. 2C) and 0.2 cm (Fig. 2D), after 7 and 21 days respectively. Patient was discharged home seven days following the wound closure (Fig. 2B). Clinical examination and ecography performed in the follow-up confirmed the successfully progressive closure of the sternal wound and the correspondent reduction of the injected MSC cluster. Patient is under normal conditions and alive after an 18 month follow-up period. Regenerative medicine has been proposed in clinical practice not only for approaching systemic disease but also to treat recurrent wound infection following cardiac surgery [3]. However, to our knowledge, the case reported here represents the first time which autologous MSCs derived from mammary artery have been used for treating recurrent mediastinal infection. Our hypothesis of MSC mechanism of action is related to their differentiation behavior in endothelial and stromal cells, such as fibroblast. On this basis we suppose that they are committed to generate and promote stromal and vessel structure generation. Thus, even preliminary, our data are in support for the development of autologous bank cell facilities dealing for cardio-tissue engineering. Conflict of interest The corresponding author confirms that the authors have no financial as well as non-financial competing interests in the manuscript.
Acknowledgments This work was partially supported by the project PON a3-00359 and by PON 04a3_00433. The authors thank Professor Enzo Di Fabrizio, Dr. Tania Limongi and Dr. Arturo Mario Minniti for their kind technical support and Dr. Gianluca Rotta, scientific responsibility of SCENIC Project. References [1] J.H. Braxton, C.A. Marrin, P.D. McGrath, J.R. Morton, M. Norotsky, D.C. Charlesworth, S.J. Lahey, R. Clough, C.S. Ross, E.M. Olmstead, G.T. O'Connor, 10-year follow-up of patients with and without mediastinitis, Semin. Thorac. Cardiovasc. Surg. 16 (1) (2004) 70–76. [2] M. Lusini, A. Di Martino, C. Spadaccio, A. Rainer, M. Chello, M. Fabbrocini, R. Barbato, V. Denaro, E. Covino, Resynthesis of sternal dehiscence with autologous bone graft and autologous platelet gel, J. Wound Care 21 (2) (2012) 74 (76–7). [3] G.F. Serraino, A. Dominijanni, F. Jiritano, M. Rossi, A. Cuda, S. Caroleo, A. Brescia, A. Renzulli, Platelet-rich plasma inside the sternotomy wound reduces the incidence of sternal wound infections, Int. Wound J. (2013) 1–5.
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N. Malara et al. / International Journal of Cardiology 182 (2015) 466–468
Fig. 2. (A) Sternal wound appeared still necrotic with no granulation tissue after 10 days of VAC therapy and i.v. infusion of antibiotic treatments. Collected autologous mesenchymal stem cells were injected just above the periosteum. (B) Final wound closure. (C) The injection points of MSCs appeared as hyper-reflective nodule areas (as indicated by red arrow) characterized by regular shape and maximum diameter of 0.88 cm (C) and 0.2 cm (D), after 7 and 21 days respectively.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Novel application of mesenchymal stem cells from mammary artery Natalia Malara a,b, Giuseppe Musolino c, Valentina Trunzo a, Arturo Mario Minniti a, Lucia Cristodoro c, Massimo Polistina c, Attilio Renzulli c, Vincenzo Mollace a,⁎ a b c
Institute of Research for Food Safety and Health (IRC-FSH), Department of Health Science University “Magna Graecia” of Catanzaro, Italy Bionem Laboratories, Department of Experimental and Clinical Medicine, Salvatore Venuta Campus, University “Magna Graecia” of Catanzaro Italy Cardiac Surgery Unit, Department of Medical and Surgical Sciences, Salvatore Venuta Campus, University “Magna Graecia” of Catanzaro Italy
a r t i c l e
i n f o
Article history: Received 30 December 2014 Accepted 2 January 2015 Available online 6 January 2015 Keywords: Autologous mesenchymal stem cells Mammary artery Mediastinitis
The incidence of 1 to 4% of mediastinitis has been reported to occur as a consequence of cardio-thoracic surgery procedures. This complication continues to have a dramatic impact on patient outcomes reporting hospital mortality between 10 and 20% [1]. Recently, vacuum therapy and new techniques as wound dressing with plasma rich of platelets and wound reconstruction with autologous or heterologous tissue [2] have improved clinical results; nevertheless morbidity remains consistent and mortality still occurs in patients with co-morbidities following prolonged hospital stay. Thus, not-conventional approaches have been proposed to get better results in regenerative medicine applied to secondary mediastinitis. Here we report on recurrent sternotomy infection refractory to conventional treatments, successfully approached with an original protocol based on the use of autologous MSC derived from fragments of left internal mammary artery (LIMA) not utilized during the first CABG operation. A 83 year old, man, suffering from type 2 diabetes mellitus treated with insulin therapy, hypochromic anemia, hypertension, peripheral artery occlusive disease, three-vessel coronary artery disease, underwent on pump CABGx3 through median sternotomy. LIMA was harvested and implanted on the left anterior descending artery. Sequential venous graft was performed on posterior descending and obtuse marginal braches. Excess of the length of mammary artery was sent to the Human Cellular Biology Laboratory IRC-FSH1 to isolate and culture endothelial cells for further characterization. Moreover, in order to ⁎ Corresponding author at: IRC-FSH, University “Magna Graecia” of Catanzaro, Campus Universitario Germaneto, 88100 Catanzaro, Italy. E-mail address: [email protected] (V. Mollace). 1 Interregional Research Center for Food Safety and Health.
http://dx.doi.org/10.1016/j.ijcard.2015.01.007 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
reduce the risk of post-operative infection sterna wound was approximated over 5 ml of autologous platelet gel. Patient informed consent was previously obtained as part of “SCENIC2” project approved by ethical committee of “Mater Domini”. Biopsied vessels were immediately immersed in standard medium with complete F-12K growth medium, 0.1 mg/ml heparin, 0.03 mg/ml endothelial cell growth supplement (ECGS), fetal calf serum 10%, gentamicin 3%, amphotericin D 3% and rinsed with phosphate-buffered saline. The vessel fragment was placed in a culture dish (Fig. 1), the adipose tissue was removed, the vessel was de-endothelialized cutting lengthwise. The remaining layer was cut into 1.5 mm pieces. Each piece was placed in a 96-well culture plate adding 50 μL of fresh F-12K complete growth medium, supplemented with 5 μL (1:10) of ECGS. Successively cells were incubated at 37 °C and 5% CO2. Cytometric evaluation allowed cell surface characterization of adult MSC. CD44positive (45%), CD45negative (72%) and fibronectinpositive (53%) expression confirmed the mesenchymal profile. Post-operative course was initially uneventfully and patient was discharged home on the tenth postoperative day. Six days following hospital discharge, patient was readmitted with fever leucocitosis, sternal instability and purulent discharge from the sternal wound. Surgical debridement was performed, sterna wires were removed, and VAC therapy was started. Bacteriologic analyses showed an infection supported by Staphylococcus epidermidis and Staphylococcus hominis. VAC therapy and i.v. antibiotic infusion were continued for six weeks. Despite of negative tissue culture results, the sternal wound appeared still necrotic with few granulation tissue. At this point we performed wound closure over subcutaneous drainage. Nevertheless, after fourteen days we observed complete wound dehiscence (Fig. 2A). We were faced with a patient in advanced age, psychologically depressed. We discussed with him about the possibility to perform an alternative therapy to improve wound healing avoiding further procedures under general anesthesia. We proposed to him to close the wound under local anesthesia, and to implant autologous MSCs isolated in order to improve tissue replacement and vascularization. Therefore, wound was stitched under local anesthesia and skin was re-approximated using 4 full thickness treads of mersilene 5/0. Collected MSCs were injected just above the periosteum with 22G needle. Three injections for each sterna side were performed. We monitored the injection points of MSCs with ultrasonography technique through a 9 MHz transducer. MSCs at injection point appeared as hyper reflective nodule areas characterized by regular shape and 2
Standardization of Circulating ENdothelIal Cells evaluation.
N. Malara et al. / International Journal of Cardiology 182 (2015) 466–468
467
Fig. 1. (A) Excess of the length of mammary artery fragment immersed in standard medium. (B) In vitro expansion human primary endothelial cells. (C) In blue the nuclear staining with DAPI. (D) Immunofluorescence in red showed the surface positivity for CD146 expression, differentiated endothelial antigen. In vitro MSCs expanded (E) and stained with DAPI (F) to evidence nuclei. Cytometric evaluation of MSC phenotype. Dot plots showed MSCs in side scattering (SSC) and forward scattering (FSC), their negativity for CD45 and positivity expression for CD44 and fibronectin antigens.
maximum diameter of 0.88 cm (Fig. 2C) and 0.2 cm (Fig. 2D), after 7 and 21 days respectively. Patient was discharged home seven days following the wound closure (Fig. 2B). Clinical examination and ecography performed in the follow-up confirmed the successfully progressive closure of the sternal wound and the correspondent reduction of the injected MSC cluster. Patient is under normal conditions and alive after an 18 month follow-up period. Regenerative medicine has been proposed in clinical practice not only for approaching systemic disease but also to treat recurrent wound infection following cardiac surgery [3]. However, to our knowledge, the case reported here represents the first time which autologous MSCs derived from mammary artery have been used for treating recurrent mediastinal infection. Our hypothesis of MSC mechanism of action is related to their differentiation behavior in endothelial and stromal cells, such as fibroblast. On this basis we suppose that they are committed to generate and promote stromal and vessel structure generation. Thus, even preliminary, our data are in support for the development of autologous bank cell facilities dealing for cardio-tissue engineering. Conflict of interest The corresponding author confirms that the authors have no financial as well as non-financial competing interests in the manuscript.
Acknowledgments This work was partially supported by the project PON a3-00359 and by PON 04a3_00433. The authors thank Professor Enzo Di Fabrizio, Dr. Tania Limongi and Dr. Arturo Mario Minniti for their kind technical support and Dr. Gianluca Rotta, scientific responsibility of SCENIC Project. References [1] J.H. Braxton, C.A. Marrin, P.D. McGrath, J.R. Morton, M. Norotsky, D.C. Charlesworth, S.J. Lahey, R. Clough, C.S. Ross, E.M. Olmstead, G.T. O'Connor, 10-year follow-up of patients with and without mediastinitis, Semin. Thorac. Cardiovasc. Surg. 16 (1) (2004) 70–76. [2] M. Lusini, A. Di Martino, C. Spadaccio, A. Rainer, M. Chello, M. Fabbrocini, R. Barbato, V. Denaro, E. Covino, Resynthesis of sternal dehiscence with autologous bone graft and autologous platelet gel, J. Wound Care 21 (2) (2012) 74 (76–7). [3] G.F. Serraino, A. Dominijanni, F. Jiritano, M. Rossi, A. Cuda, S. Caroleo, A. Brescia, A. Renzulli, Platelet-rich plasma inside the sternotomy wound reduces the incidence of sternal wound infections, Int. Wound J. (2013) 1–5.
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N. Malara et al. / International Journal of Cardiology 182 (2015) 466–468
Fig. 2. (A) Sternal wound appeared still necrotic with no granulation tissue after 10 days of VAC therapy and i.v. infusion of antibiotic treatments. Collected autologous mesenchymal stem cells were injected just above the periosteum. (B) Final wound closure. (C) The injection points of MSCs appeared as hyper-reflective nodule areas (as indicated by red arrow) characterized by regular shape and maximum diameter of 0.88 cm (C) and 0.2 cm (D), after 7 and 21 days respectively.
