Original article
Adipose-derived mesenchymal stem cells in biosutures do not improve healing of experimental colonic anastomoses 5−7 ´ ´ I. Pascual1−3 , G. Fernandez de Miguel4 , U. A. Gomez-Pinedo , F. de Miguel2 , M. Garc´ıa Arranz2 1−3 and D. Garc´ıa-Olmo 1
Department of General Surgery and 2 Cell Therapy Unit, La Paz University Hospital, Madrid, 3 Department of Surgery, Autonoma University of ˜ ´ Pr´ıncipe Felipe, Valencia, Spain, and 6 Centro de Investigacion ´ y Asistencia en Tecnolog´ıa y Diseno Madrid, 4 Cellerix, Madrid, 5 Centro de Investigacion ´ del Estado de Jalisco/Consejo Nacional de Ciencia y Tecnolog´ıa and 7 Organismo Publico Descentralizado Hospital Civil de Guadalajara, Guadalajara, Mexico Correspondence to: Dr I. Pascual, Servicio de Cirug´ıa General C, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046, Madrid, Spain (e-mail:
[email protected])
Background: The feasibility of producing ‘biosutures’ coated with adipose-derived mesenchymal stem
cells (ASCs) was assessed and their effect on colonic anastomoses evaluated. Methods: Sutures were cultured with ASCs isolated from BDIX rats before analysis by light and scanning electron microscopy. Anastomoses were performed in 40 BDIX rats using either biosutures or conventional sutures. Histopathological features, adhesion formation and anastomotic strength were evaluated. Results: Sutures were coated with ASCs within 24 h. Anastomoses made with biosutures had a lower adhesion index only during the first week (P = 0·006 at 7 days), but not subsequently. There were no significant differences in anastomotic healing with the two types of suture. Conclusion: ASC biosutures have no effect on anastomotic healing in rats. Presented to the XXVIth National Congress of Surgery, Madrid, Spain, November 2006 Paper accepted 26 March 2008 Published online in Wiley InterScience (www.bjs.co.uk). DOI: 10.1002/bjs.6242
Introduction
Adipose cells
The rate of healing of colorectal anastomoses is dependent on patient variables, technical factors and cellular interactions1 . Pluripotent mesenchymal stem cells (MSCs) from several sites, including bone marrow and adipose tissue2 , may have inmunomodulatory properties3 , indicating a possible use for MSCs in regenerative medicine and tissue engineering4 . This study assessed the use of sutures coated with adipose-derived stem cells (ASCs) (‘biosutures’) on experimental healing of colonic anastomoses.
Adipose cells were isolated using a method described for human adipose tissue5 . Briefly, subcutaneous fat was removed from two male BDIX rats, washed with sterile phosphate-buffered saline (PBS) and digested with type I collagenase (0·075 per cent; Gibco, Paisley, UK) for 30 min at 37° C to release the cellular fraction. Cells were resuspended in medium, then plated in culture dishes at a concentration of 2–3 × 104 cells/cm2 . Subconfluent cells were transfected with copepod green fluorescent protein (copGFP) using retroviral particles according to TM the manufacturer’s instructions (Retrofect kit; Genetrix, Madrid, Spain) (Fig. 1a,b).
Methods
Forty-two syngeneic BDIX rats (weighing 250–360 g) were kept under standardized conditions. The study was approved by the Institutional Committee on the Ethical Use of Animals for Experimentation. Copyright 2008 British Journal of Surgery Society Ltd Published by John Wiley & Sons Ltd
Preparation of biosutures TM
Braided polyglactin 910 sutures (Vicryl ; Ethicon, Edinburgh, UK) were cut into lengths of 30 cm and placed in ultra-low attachment six-well plates (Costar, Corning, British Journal of Surgery 2008; 95: 1180–1184
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ASCs in vitro − bright field
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ASCs in vitro − fluorescence
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ASCs on monofilament suture
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ASCs on multifilament suture
a Bright-field and b fluorescent images of adipose-derived mesenchymal stem cells (ASCs) in culture; copepod green fluorescent protein (copGFP) is expressed in the majority of cells. c Monofilament and d braided multifilament absorbable sutures coated with copGFP-labelled ASCs under fluorescent light
Fig. 1
New York, USA) with PBS. Subconfluent copGFPlabelled cells were collected and resuspended in culture medium at 750 000 cells/ml; after removal of PBS, 2 ml of this cell suspension was added to the sutures. Plates with the cell suspension in contact with the sutures were placed in an incubator at 37° C in an atmosphere of 5 per cent carbon dioxide in air with gentle agitation. After 24 h, the culture medium and non-adherent cells were removed and fresh medium was added. Sutures were maintained in the incubator under the same conditions until use on the same day. For simplification, all animal experiments were performed only with multifilament braided sutures. Preliminary experiments showed that copGFP-labelled ASCs Copyright 2008 British Journal of Surgery Society Ltd Published by John Wiley & Sons Ltd
detached from the biosuture after two passes through muscular tissue, so a single thread of either control suture or biosuture was used for only two stitches.
Anastomosis model Anaesthesia was induced with a mixture of ketamine (100 mg/kg), atropine (0·4 mg/kg) and diazepam (8 mg/kg) by intraperitoneal injection. A 3-cm midline laparotomy was performed. The colon was sectioned completely 5 cm from the ileocaecal valve, and a colonic end-to-end anastomosis was constructed with six single interrupted stitches using either control sutures or biosutures. Finally, www.bjs.co.uk
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the abdominal wall was closed in two layers using 2/0 silk sutures.
Experimental design The 40 remaining rats were divided into four groups. In each group of ten animals, five colonic anastomoses were performed with control sutures (polyglactin 910) and five with biosutures. Anastomoses were examined 4, 7, 14 and 21 days later. The study was blinded during surgery and analysis of anastomoses. Six variables were analysed when the abdomen was opened: dehiscence, dilatation, obstruction, general adhesions, separation of adhesions and adherent structures6 . An adhesion index was calculated as the sum of the general Control suture Biosuture
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Bursting pressure (mm Hg)
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adhesions index plus the index of separation of adhesions plus the number of adherent structures. Intraluminal anastomotic pressure was monitored by a pressure sensor connected to a computer with specific software (Polygram 98; Medtronic, Shoreview, Minnesota, USA) while injecting saline containing methylene blue into the intestinal segment. The maximum pressure before bursting (the point at which leakage was noted at any site) was recorded as the bursting pressure7 . Bursting tension was calculated using Laplace’s law, in order to identify differences in resistance between rat tissues of different sizes8 . After recording the bursting pressure, the intestinal segment was fixed immediately in 4 per cent formaldehyde. Once the internal perimeter of the anastomosis
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Fig. 2 a Adhesion index, b bursting pressure, c radius and d bursting tension of colonic anastomoses performed with control sutures and biosutures. Values are mean(s.d.). * P = 0·025, †P = 0·006 (Mann–Whitney U test)
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British Journal of Surgery 2008; 95: 1180–1184
Biosutures and anastomotic healing
had been measured with calipers (for determination of the anastomotic radius), the tissue was processed for histological examination, using haematoxylin and eosin stain to assess inflammation (light, moderate or intense), presence of granulomas (few or abundant) and fibrosis.
Statistical analysis The χ2 test was used to compare dehiscence, dilatation, obstruction, inflammation, granulomas and fibrosis within and between the groups. The Mann–Whitney U test was employed to compare the adhesion index, bursting pressure, bursting tension and anastomotic radius. P < 0·050 was considered statistically significant.
Results
The use of ultra-low attachment culture plates favoured the attachment of ASCs to the only surface available in the wells, the suture. ASCs adhered to both monofilament and multifilament sutures within the first few hours, coating the threads almost completely after 24 h (Fig. 1c,d). The use of biosutures did not affect the incidence of dehiscence, dilatation, obstruction or pattern of inflammation at any timepoint compared with control sutures (data not shown). The adhesion index was significantly lower in the biosuture group at days 4 (P = 0·025) and 7 (P = 0·006), but not at later times (Fig. 2a). There were no differences between the groups in bursting pressure, radius and bursting tension (Fig. 2b–d).
Discussion
This study has demonstrated the feasibility of producing sutures covered with stem cells (called ‘biosutures’). Such material should allow regenerative therapy to reach the site at which healing occurs. A lower adhesion index was found only during the first few days and might be due to an immunomodulatory effect of stem cells, which suppress the in vitro proliferation of activated lymphocytes3,9 . In a rat model of injured mesothelium10 , the adhesion score was decreased when MSCs were injected intraperitoneally immediately after surgery. This decrease was attributed to the ability of MSCs to differentiate into mesothelial cells capable of repopulating the peritoneum10 . Adipose tissue- and bone marrowderived MSCs differentiate into cells of mesodermal Copyright 2008 British Journal of Surgery Society Ltd Published by John Wiley & Sons Ltd
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origin, but less is known about differentiation into nonmesodermal cells11 . The present histological study could not confirm differentiation of ASCs into other cell types. The biosutures were coated with stem cells, but the study demonstrated no apparent advantage for these sutures compared with conventional sutures. Acknowledgements
The authors thank Jos´e Manuel Garc´ıa-Verdugo, Cen´ tro de Investigacion Pr´ıncipe Felipe and Instituto Cavanilles, University of Valencia, Spain, for assistance with microscopy of biosutures, and Dr Pilar Castillo, Digestive Consultant, La Paz University Hospital, Madrid, Spain, for help with the bursting pressure experiments. This work was supported in part by funds from Cellerix to the Chair of Cell Therapy and Regenerative Medicine, Autonoma University of Madrid. References 1 Thompson SK, Chang EY, Jobe BA. Clinical review: healing in gastrointestinal anastomoses, part I. Microsurgery 2006; 26: 131–136. 2 Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13: 4279–4295. 3 Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Br J Haematol 2005; 129: 118–129. 4 Fraser JK, Wulur I, Alfonso Z, Hedrick MH. Fat tissue: an underappreciated source of stem cells for biotechnology. Trends Biotechnol 2006; 24: 150–154. 5 Garc´ıa-Olmo D, Garc´ıa-Arranz M, Herreros MD, Pascual I, Peiro C, Rodr´ıguez-Montes JA. A phase I clinical trial of the treatment of Crohn’s fistula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum 2005; 48: 1416–1423. 6 Garc´ıa-Olmo D, Lucas FJ, Paya J. Relationship between peritoneum adhesion phenomena and the experimental resistance of colon anastomoses: influence of omentoplasty. Eur Surg Res 1996; 28: 315–322. 7 Garc´ıa-Olmo D, Pay´a J, Lucas FJ, Garc´ıa-Olmo DC. The effects of the pharmacological manipulation of postoperative intestinal motility on colonic anastomosis. An experimental study in a rat model. Int J Colorectal Dis 1997; 12: 73–77. 8 Garc´ıa-Olmo DC, Pay´a J, Garc´ıa-Olmo D. Effects of perioperative treatment with TNP-470 on the resistance of colonic anastomoses in rats. Dig Surg 2000; 17: 154–159. 9 Le Blanc K. Inmunomodulatory effects of fetal and adult mesenchymal stem cells. Cytotherapy 2003; 5: 485–489.
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10 Lucas PA, Warejcka DJ, Zhang LM, Newman WH, Young HE. Effect of rat mesenchymal stem cells on development of abdominal adhesions after surgery. J Surg Res 1996; 62: 229–232.
¨ 11 Sch¨affler A, Buchler C. Concise review: adipose tissue-derived stromal cells – basic and clinical implications for novel cell-based therapies. Stem Cells 2007; 25: 818–827.
Peripheral schwannoma A 60-year old man presented with a slow growing antebrachial tumour. Digital pressure induced paresthesia in the superficial radial nerve dermatome (Tinel’s sign). Excision was performed by an interfascicular approach, after visualizing the proximal and distal nerve. Histological examination continued a benign schwannoma. Postoperative function and sensitivity were uncompromised.
MJA Loos, M.D., RMH Roumen, M.D., PhD: Department of General Surgery M´axima Medical Centre PO Box 7777 De Run 4600 5500 MB Veldhoven The Netherlands Tel +31 40 888 8550 Fax +31 40 888 8565 (e-mail:
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