Journal of Pediatric Surgery xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Extraluminal distraction enterogenesis using shape-memory polymer Jeremy G. Fisher a,b, Eric A. Sparks a,b, Faraz A. Khan a,b, Beatrice Dionigi b, Hao Wu c, Joseph Brazzo III b, Dario Fauza b, Biren Modi a,b, David L. Safranski d, Tom Jaksic a,b,⁎ a
Center for Advanced Intestinal Rehabilitation (CAIR), Boston Children's Hospital and Harvard Medical School, Boston, MA Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA d MedShape, Inc., Atlanta, GA b c
a r t i c l e
i n f o
Article history: Received 2 March 2015 Accepted 10 March 2015 Available online xxxx Key words: Intestinal failure Citrulline Short bowel syndrome Biological markers Intestinal adaptation
a b s t r a c t Purpose: Although a few techniques for lengthening intestine by mechanical stretch have been described, they are relatively complex, and the majority involve placement of an intraluminal device. Ideally, techniques applicable to humans would be easy to perform and extraluminal to avoid the potential for mucosal injury. This study of distraction enterogenesis used an extraluminal, radially self-expanding shape-memory polymer cylinder and a simple operative approach to both elongate intestine and grow new tissue. Methods: Young Sprague Dawley rats (250–350 g) underwent Roux-en-Y isolation of a small intestinal limb and were divided in three groups: no further manipulation (Control 1, C1); placement of a nonexpanding device (Control 2, C2); or placement of a radially expanding device by the limb (Experimental, Exp). For C2 and Exp animals, the blind end of the limb was wrapped around the radially expanding cylindrical device with the limb-end sutured back to the limb-side. Bowel length was measured at operation and at necropsy (14 days) both in-situ and ex-vivo under standard tension (6 g weight). Change in length is shown as mean ± standard deviation. A blinded gastrointestinal pathologist reviewed histology and recorded multiple measures of intestinal adaptation. The DNA to protein ratio was quantified as a surrogate for cellular proliferation. Changes in length, histologic measures, and DNA:protein were compared using analysis of variance, with significance set at P b 0.05. Results: The length of the Roux limb in situ increased significantly in Exp animals (n = 8, 29.0 ± 5.8 mm) compared with C1 animals (n = 5, −11.2 ± 9.0 mm, P b 0.01). The length of the Roux limb ex vivo under standard tension increased in the Exp group (25.8 ± 4.2 mm) compared with the C2 group (n = 6, −4.3 ± 6.0, P b 0.01). There were no differences in histologic measures of bowel adaptation between the groups, namely villous height and width, crypt depth, crypt density, and crypt fission rate (all P ≥ 0.08). Muscularis mucosal thickness was also not different (P = 0.25). There was no difference in DNA:protein between groups (P = 0.47). Conclusion: An extraluminally placed, radially expanding shape-memory polymer cylinder successfully lengthened intestine, without damaging mucosa. Lack of difference in muscularis thickness and a constant DNA:protein ratio suggests that this process may be related to actual growth rather than mere stretch. This study demonstrated a simple approach that warrants further study aiming at potential clinical applicability. © 2015 Elsevier Inc. All rights reserved.
1. Background Intestinal failure (IF) is an intrinsic bowel disease that results in the inability to sustain growth or maintain electrolyte and fluid homeostasis [1]. Medical advances, such as the development of intensive interdisciplinary intestinal rehabilitation programs have improved outcomes [2]. Though autologous intestinal reconstructive surgeries (AIRS) like longitudinal intestinal lengthening and tapering (LILT) [3] and serial transverse enteroplasty (STEP) [4] can improve bowel function, their use is limited to patients with significant intestinal dilation. ⁎ Corresponding author at: Center for Advanced Intestinal Rehabilitation (CAIR), Boston Children's Hospital, 300 Longwood Avenue, Fegan 3, Boston, MA, 02115. Tel.: +1 617 355 9600; fax: +1 617 730 0477. E-mail address: [email protected] (T. Jaksic).
Mechanical stretch has been used for many years to stimulate growth in tissues such as bone and skin [5,6]. Experimentally, traction on bowel has been shown to produce additional tissue (enterogenesis) and a variety of devices and operative approaches have been used in animals, but none of these strategies have yet translated into clinical use [7–12]. Most of these devices must be placed within the lumen of the bowel, necessitating violation of the bowel wall, creation of a closed loop, and potentially damaging the mucosa. Some require complex activation mechanisms that require external activation. Further, none of the reported devices are FDA cleared or directly predicated on a cleared device. Shape-memory polymers (SMPs) are plastics that have the ability to change configuration when activated [13]. SMPs can be produced in essentially any geometry, may be heat-activated, and recover their shape with relatively constant velocity, the degree of which can be specified
http://dx.doi.org/10.1016/j.jpedsurg.2015.03.013 0022-3468/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
2
J.G. Fisher et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
by the designer [14–16]. Currently, there are FDA-cleared devices using SMPs, and they are indicated for orthopedic use. This study used a radially self-expanding SMP device placed extraluminally using a simple operative approach to produce distraction enterogenesis in a rat model. The specific aims were to: (1) demonstrate elongation of intestine and (2) determine whether such elongation was a product of true tissue growth using histologic and biochemical markers.
2.3. Nutrition
2. Methods
2.4. Measurements
The Institutional Animal Care and Use Committee at Boston Children's Hospital approved the study (#13-01-2356). Adolescent Sprague–Dawley rats were assigned to one of three groups: a control group without device placement (Control 1, C1), a control group with nonexpanding device placement (Control 2, C2), or the experimental group in which an expanding device was placed (Experimental, Exp).
The length of the Roux limb was measured off tension in duplicate along the antimesenteric border in situ at operation and again at necropsy. The portion that was wrapped around the device was measured in a similar fashion. At necropsy, a standard 6 g weight was hung from one end of the Roux limb and the length of the whole limb and wrapped (experimental) segment were each measured (tension).
All animals ate a standard solid chow diet until 24 hours before operation, during which they were provided a liquid diet ad libitum (Bio-Serv, Frenchtown, NJ). Liquid diet was continued postoperatively until the animals could tolerate at least 100 mL per day and had passed stool, at which time standard solid chow was restarted.
2.1. Operative procedures 2.5. Histologic evaluation In all animals, a midline laparotomy was performed. Isolated, Roux-enY segments were created in each animal: the bowel was the eviscerated and a location approximately 30 cm from the ligament of Treitz (LOT) was selected for transection. An anastomosis was then created between the proximal cut end and a location 6–10 cm from the distal cut end [single layer, interrupted 6-0 PDS (Ethicon, Somerville, NJ)], leaving a length of isolated small intestine (the Roux limb). For C1 animals, the blind end of the Roux limb was ligated and the abdomen was closed with no further intervention. In both C2 and Exp animals, the blind end was wrapped around a coiled, cylindrical shape-memory polymer device. The blind end was then closed and sutured to the side of the roux limb (Figs. 1 and 2). In the C2 animals, this device did not expand, while in Exp animals, the device expanded radially (Fig. 2).
At necropsy, a segment of small bowel from the Roux limb (control 1) or wrapped segment (control 2, experimental) were preserved in 4% formalin. Care was taken to avoid areas adjacent to sites of anastomosis. They were then embedded for frozen section, sectioned, stained with hematoxylin and eosin, and reviewed by a gastrointestinal pathologist who was blinded to operative group. Mucosal thickness/crypt depth, thickness of internal muscularis propria (the circular layer), villous height and width were measured in each sample with cellSens digital imaging software (Olympus, USA) according to manufacturer's instruction. The parameters were evaluated in 10 distinct sites and the average was recorded. Crypt density and fission rate were determined using previously validated methodology [17].
2.2. Device 2.6. Total DNA and protein A sheet of SMP was rolled into a cylinder, which was designed to expand radially over 7 days when activated by body heat and moisture. To create the polymer, isobornyl acrylate, 2-hydroxyethylacrylate, and 1,6-hexanediol diacrylate were mixed in weight ratio of 75:20:5, respectively. 0.5 wt% of phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide was used as a photoinitiator. All reagents were used as received (Sigma-Aldrich, St. Louis, MO). The mixture was injected into a glass mold with a 0.86 mm spacer and photopolymerized for 3 minutes at 405 nm using a LED flood lamp. After mold removal, the polymer was cut with a CO2 laser (Gravograph LS-500, Gravotech, Inc, Duluth, GA) into 15 mm × 47 mm rectangles with rounded edges. In order to program the temporary shape, the thin rectangles were wrapped around a mandrel in 80 °C water to form a cylinder with a diameter of 8.5 mm and height of 15 mm. They were constrained with plastic ties while cooling to store the temporary shape.
Measurements of total DNA and protein contents in the isolated bowel segment wall were performed in snap-frozen fresh samples based on methods as previously described [18]. Briefly, DNA and protein were isolated using the All Prep DNA/RNA/Protein Mini Kit (Qiagen, Gaithersburg, MD) per manufacturer's instructions. DNA was quantified using a NanoDrop 8000 (Thermo Scientific, Waltham, MA), with nucleic acid concentrations reported as mg/mL. Total protein was determined using the colorimetric DC Protein Assay (BioRad, Hercules, CA) per manufacturer's instructions, based on the reaction of protein with an alkaline copper tartrate solution and Folin reagent. Absorbances were read at 750 nm on a microplate reader (FLUOStar Omega; BMG Labtech, Cary, NC). A standard curve was obtained using known concentrations of bovine serum albumin provided by the manufacturer. Protein concentrations were also reported as mg/mL.
Fig. 1. Schematic for experimental animals; creation of Roux-en-Y limb, placement of cylindrical coil and radial expansion (over 7 days).
Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
J.G. Fisher et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
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Fig. 2. Photographs at operation. Radial expanding shape memory polymer device (A) placed with end of Roux limb (B) wrapped around it (C and D). Control group 2 underwent the same operation with placement of a nonexpanding polymer coil.
2.7. Statistical analysis
4. Discussion
Animals surviving to 14 days were included. Analysis was performed using JMP Pro version 10.0 (SAS Institute, Cary, NC). Change in length, percent change in length, all histologic measures, and DNA:protein were all compared using Student's t-test. All values are presented as mean ± standard deviation.
Distraction enterogenesis, or creating intestinal tissue by mechanical stretch, is a promising new therapy for short bowel syndrome (SBS). The applied tension induces growth via several known molecular mechanotransduction pathways [19], stimulates mesenteric neovascularization [20], and stretched jejunal segments appear to function well when replaced into continuity [21]. Despite the number of devices now described in the literature, none has yet translated into clinical practice. The extant approaches are relatively complex and have significant limitations, such as the need to violate the bowel wall, the use of a closed loop, or the need for a complex activating mechanism [7–12]. This investigation demonstrates that a self-expanding polymer placed in a simple extraluminal configuration can elongate small bowel and appears to induce growth. The percent change in length was significantly higher in the experimental group compared with the device control. Because only a short portion of the long Roux limb was stretched, the lack of difference in Roux length is not surprising (a ~2 cm portion of the ~10 cm limb was stretched by an additional ~2 cm, a large change relative to the experimental segment but a small change relative to the whole limb). The extraluminal approach used here doubled the length of the target segment on average, which is a similar degree of increase to other techniques [7–12]. To produce this degree of elongation, the device only increased in diameter by 70%, substantially less than those used in other approaches. The intraluminal devices used previously produce tension by expanding longitudinally along the axis of the bowel. This configuration requires that the device expand as much or more than the intestine is expected to stretch (~ 1:1 ratio). By wrapping the bowel around a radially-expanding device, it stretches with the circumference of the implant, meaning that each increase of 1 mm in device diameter produces 3.14 (or 1 × π) mm in bowel stretch. Thus this design potentially allows for a greater increase in bowel length for each increment of device expansion. A more compact device will be advantageous in patients where a substantial degree of growth is needed, but the dimensions of the abdominal cavity limit the maximum implant size. It is possible that in the future this advantage may be further multiplied by wrapping multiple loops around the device. The lack of difference between groups in both muscularis thickness and DNA:protein ratio suggests that the elongation seen is reflective of actual tissue growth rather than simple stretch with thinning of tissue. Though evaluating markers of cellular proliferation potentially may have enhanced such an analysis, this approach may have been confounded in our model by the presence of fibrotic tissue that developed in proximity to the device. Future studies will utilize techniques that can appropriately delineate histologic types and measure proliferation in intestinal tissue [22]. No differences were seen among histologic markers for intestinal adaptation such as villus height, crypt depth, and crypt fission rate. However, the distal portion of the Roux limb that was stretched was remote from the flow of luminal nutrients, which is an important stimulus for typical mucosal adaptation [23],
3. Results The overall survival rate was 73% (19/26). Deaths occurred in each group and were attributed to stricture or leak at the Roux-en-Y anastomosis or complications of anesthesia. There were 5 animals surviving to 14 days in the C1 group, 6 in C2, and 8 in Exp. Weight gain was similar between groups (P = 0.68). Bowel measurements at operation (day 0) and necropsy (day 14) are shown in Table 1. The Roux limb length of the two control groups C1 and C2 were measured in situ (0 ± 0% vs 20 ± 46%, P = 0.06) and under standard tension (0 ± 21% vs 3 ± 34%, p = 0.37) at necropsy, demonstrating no change in length. SMP devices in the Exp group increased from 28 ± 2 to 47 ± 10 mm in circumference (70% increase). Experimental segments increased from 24 ± 3 mm to 50 ± 15 mm in the Exp group and 35 ± 9 mm to 37 ± 6 mm in the C2 group when measured on standard tension (percent change 107 ± 52% vs 7 ± 31%, P = 0.001). There was a variable amount of fibrosis around the device and experimental segment in both Exp and C2 groups. These adhesions were removed after in situ measurements were made. There were no significant differences in villus height, width, muscularis thickness, crypt density, or crypt fission rate between the three groups (Fig. 3). Crypts were less deep in the C1 group than C2 or Exp (P = 0.02). The total DNA masses in each group were: C1 0.015 ± 0.017, C2 0.008 ± 0.008, Exp 0.006 ± 0.003 mg. Total protein masses were: C1 0.33 ± 0.05, C2 0.27 ± 0.03, Exp 0.29 ± 0.06 mg. There were no differences in DNA:protein between groups (Fig. 4, P = 0.41).
Table 1 Lengths of wrapped bowel segments at operation and necropsy (day 14). Measurements were made on the antimesenteric border in situ and under standard (6 g) tension ex vivo (at necropsy). Wrapped Segment Length: Control (C2) Change (mm) In situ Tension Percent change In situ Tension
Experimental
p
11 ± 7 1 ± 10
28 ± 10 26 ± 13
0.003⁎ 0.002⁎
29 ± 20 7 ± 31
114 ± 31 107 ± 52
b0.001⁎ 0.001⁎
⁎ P b 0.05, Student's t-test.
Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
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J.G. Fisher et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
Fig. 3. Histologic measures. There were no differences between groups except in crypt depth: the crypts in the Control 1 group were less deep than the other two groups (*P = 0.02). All comparisons by ANOVA.
and thus a lack of difference may not be surprising. Further study using this technique to stretch bowel that remains in continuity is needed to clarify the effect of stretch on mucosal adaptation. This model has a number of advantages over previously studied approaches. Shape-memory polymers can expand when activated by body heat and water uptake and will expand at a relatively constant rate determined by the polymer's chemical design. This obviates the need for external activation and thus additional hardware. Compared with an expander that is incrementally enlarged, the consistent tension applied here creates a constant stimulus for growth and hence may be less likely to tear tissue. This device is also easily scalable to a large animal model for eventual human use. The authors anticipate that a loop of small intestine in continuity could be wrapped around a similar radially expanding cylindrical device and thus a potential future advantage is that this operative approach would not require extensive dissection, violation of the intestinal wall, or the creation of a closed loop, which are likely to cause pain and potentially other complications. Further, though none of the devices used to stretch bowel are FDA approved, an orthopedic device based on a methacrylate SMP has been FDA cleared [24] and the acrylic SMP used here has a chemistry similar to other plastics used in medical applications [25,26]. The small number of animals studied and the inherent variability in measuring intestinal length limit this investigation. Further, the devices expanded somewhat less than expected and thus less of an increase in length was found than initially planned. By altering the mechanochemical properties of the polymer, however, the force applied can be increased to allow for full deployment and preliminary results with updated polymers are promising. The variable fibrosis around the devices is incompletely explained by this study and may have limited expansion to some degree. Though the potential for serosal tears exists, none of the animals were found to have died of a perforation at the wrap site. Lastly, for this initial investigation, the stretched segments were not replaced
into continuity to test their function. The authors anticipate that this technology can be easily applied to a large animal model in which the function of the resulting stretched bowel could be rigorously examined. This initial series demonstrates the feasibility of an extraluminal model for distraction enterogenesis using a shape-memory polymer device that is easily installed, self-expanding, and scalable. It successfully produced elongation of intestine that appears attributable to growth. Extraluminal, radial expansion has the potential to provide more bowel stretch in a smaller space without the need to violate the intestinal wall or expose the mucosa to a foreign body. Further study to optimize this technique for potential human use is warranted. Acknowledgments The authors would like to hank Azra Ahmed, BS for technical assistance. Appendix A. Discussion Presented by Jeremy Fisher, Boston, MA Discussant: DR. JOSH HORVATH, Los Angeles, CA I'm wondering in your self-expanding polymer, how long does it take to deploy when exposed to body heat and how do you prevent breaking the suture line open? Response: DR. FISHER The nice thing about these polymers is that you can set them by use of the chemistry. You can determine exactly how long you want them to take to resume their initial confirmation. We program these to change back to their initial shape in a week. How do you know it's not going to hurt your suture line? I think that's a little bit of trial and error. That's certainly something we were worried about here. But in this group, the tension is very low, and we did not actually see disruption of the suture line at all. I think that's one of the things we can work out as we expand to a large animal model, but it seems promising in this experiment.
References
Fig. 4. Mean DNA to protein ratio by group. Error bars represent standard deviation. P = 0.41 (ANOVA).
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Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
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[17] Dekaney CM, Fong JJ, Rigby RJ, et al. Expansion of intestinal stem cells associated with long-term adaptation following ileocecal resection in mice. Am J Physiol Gastrointest Liver Physiol 2007;293:G1013–22. [18] Tolosa JM, Schjenken JE, Civiti TD, et al. Column-based method to simultaneously extract DNA, RNA, and proteins from the same sample. Biotechniques 2007;43:799–804. [19] Sueyoshi R, Woods-Ignatoski KM, Okawada M, et al. Distraction-induced intestinal growth: the role of mechanotransduction mechanisms in a mouse model of short bowel syndrome. Tissue Eng 2014;20(3–4):830–41. [20] Ralls MW, Sueyoshi R, Herman RS, et al. Mesenteric neovascularization with distractioninduced intestinal growth: enterogenesis. Pediatr Surg Int 2013;29:33–9. [21] Stark R, Zupekan T, Bondada S, et al. Restoration of mechanically lengthened jejunum into intestinal continuity in rats. J Pediatr Surg 2011;46:2321–6. [22] Ohta Y, Ichimura K. Proliferation markers, proliferating cell nuclear antigen, Ki67, 5bromo-2'-deoxyuridine, and cyclin D1 in mouse olfactory epithelium. Ann Otol Rhinol Laryngol 2000;109:1046–8. [23] Dahly EM, Gillingham MB, Guo Z, et al. Role of luminal nutrients and endogenous GLP-2 in intestinal adaptation to mid-small bowel resection. Am J Physiol Gastrointest Liver Physiol 2003;284:G670–82. [24] MedShape Solutions. FDA 510(k) Summary. K083792. http://www.accessdata.fda. gov/cdrh_docs/pdf8/K083792.pdf. [25] Richard RE, Schwarz M, Ranade S, et al. Evaluation of acrylate-based block copolymers prepared by atom transfer radical polymerization as matrices for paclitaxel delivery from coronary stents. Biomacromolecules 2005;6:3410–8. [26] Simon D, Ware T, Marcotte R, et al. A comparison of polymer substrates for photolithographic processing of flexible bioelectronics. Biomed Microdevices 2013;15:925–39.
Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Extraluminal distraction enterogenesis using shape-memory polymer Jeremy G. Fisher a,b, Eric A. Sparks a,b, Faraz A. Khan a,b, Beatrice Dionigi b, Hao Wu c, Joseph Brazzo III b, Dario Fauza b, Biren Modi a,b, David L. Safranski d, Tom Jaksic a,b,⁎ a
Center for Advanced Intestinal Rehabilitation (CAIR), Boston Children's Hospital and Harvard Medical School, Boston, MA Department of Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA d MedShape, Inc., Atlanta, GA b c
a r t i c l e
i n f o
Article history: Received 2 March 2015 Accepted 10 March 2015 Available online xxxx Key words: Intestinal failure Citrulline Short bowel syndrome Biological markers Intestinal adaptation
a b s t r a c t Purpose: Although a few techniques for lengthening intestine by mechanical stretch have been described, they are relatively complex, and the majority involve placement of an intraluminal device. Ideally, techniques applicable to humans would be easy to perform and extraluminal to avoid the potential for mucosal injury. This study of distraction enterogenesis used an extraluminal, radially self-expanding shape-memory polymer cylinder and a simple operative approach to both elongate intestine and grow new tissue. Methods: Young Sprague Dawley rats (250–350 g) underwent Roux-en-Y isolation of a small intestinal limb and were divided in three groups: no further manipulation (Control 1, C1); placement of a nonexpanding device (Control 2, C2); or placement of a radially expanding device by the limb (Experimental, Exp). For C2 and Exp animals, the blind end of the limb was wrapped around the radially expanding cylindrical device with the limb-end sutured back to the limb-side. Bowel length was measured at operation and at necropsy (14 days) both in-situ and ex-vivo under standard tension (6 g weight). Change in length is shown as mean ± standard deviation. A blinded gastrointestinal pathologist reviewed histology and recorded multiple measures of intestinal adaptation. The DNA to protein ratio was quantified as a surrogate for cellular proliferation. Changes in length, histologic measures, and DNA:protein were compared using analysis of variance, with significance set at P b 0.05. Results: The length of the Roux limb in situ increased significantly in Exp animals (n = 8, 29.0 ± 5.8 mm) compared with C1 animals (n = 5, −11.2 ± 9.0 mm, P b 0.01). The length of the Roux limb ex vivo under standard tension increased in the Exp group (25.8 ± 4.2 mm) compared with the C2 group (n = 6, −4.3 ± 6.0, P b 0.01). There were no differences in histologic measures of bowel adaptation between the groups, namely villous height and width, crypt depth, crypt density, and crypt fission rate (all P ≥ 0.08). Muscularis mucosal thickness was also not different (P = 0.25). There was no difference in DNA:protein between groups (P = 0.47). Conclusion: An extraluminally placed, radially expanding shape-memory polymer cylinder successfully lengthened intestine, without damaging mucosa. Lack of difference in muscularis thickness and a constant DNA:protein ratio suggests that this process may be related to actual growth rather than mere stretch. This study demonstrated a simple approach that warrants further study aiming at potential clinical applicability. © 2015 Elsevier Inc. All rights reserved.
1. Background Intestinal failure (IF) is an intrinsic bowel disease that results in the inability to sustain growth or maintain electrolyte and fluid homeostasis [1]. Medical advances, such as the development of intensive interdisciplinary intestinal rehabilitation programs have improved outcomes [2]. Though autologous intestinal reconstructive surgeries (AIRS) like longitudinal intestinal lengthening and tapering (LILT) [3] and serial transverse enteroplasty (STEP) [4] can improve bowel function, their use is limited to patients with significant intestinal dilation. ⁎ Corresponding author at: Center for Advanced Intestinal Rehabilitation (CAIR), Boston Children's Hospital, 300 Longwood Avenue, Fegan 3, Boston, MA, 02115. Tel.: +1 617 355 9600; fax: +1 617 730 0477. E-mail address: [email protected] (T. Jaksic).
Mechanical stretch has been used for many years to stimulate growth in tissues such as bone and skin [5,6]. Experimentally, traction on bowel has been shown to produce additional tissue (enterogenesis) and a variety of devices and operative approaches have been used in animals, but none of these strategies have yet translated into clinical use [7–12]. Most of these devices must be placed within the lumen of the bowel, necessitating violation of the bowel wall, creation of a closed loop, and potentially damaging the mucosa. Some require complex activation mechanisms that require external activation. Further, none of the reported devices are FDA cleared or directly predicated on a cleared device. Shape-memory polymers (SMPs) are plastics that have the ability to change configuration when activated [13]. SMPs can be produced in essentially any geometry, may be heat-activated, and recover their shape with relatively constant velocity, the degree of which can be specified
http://dx.doi.org/10.1016/j.jpedsurg.2015.03.013 0022-3468/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
2
J.G. Fisher et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
by the designer [14–16]. Currently, there are FDA-cleared devices using SMPs, and they are indicated for orthopedic use. This study used a radially self-expanding SMP device placed extraluminally using a simple operative approach to produce distraction enterogenesis in a rat model. The specific aims were to: (1) demonstrate elongation of intestine and (2) determine whether such elongation was a product of true tissue growth using histologic and biochemical markers.
2.3. Nutrition
2. Methods
2.4. Measurements
The Institutional Animal Care and Use Committee at Boston Children's Hospital approved the study (#13-01-2356). Adolescent Sprague–Dawley rats were assigned to one of three groups: a control group without device placement (Control 1, C1), a control group with nonexpanding device placement (Control 2, C2), or the experimental group in which an expanding device was placed (Experimental, Exp).
The length of the Roux limb was measured off tension in duplicate along the antimesenteric border in situ at operation and again at necropsy. The portion that was wrapped around the device was measured in a similar fashion. At necropsy, a standard 6 g weight was hung from one end of the Roux limb and the length of the whole limb and wrapped (experimental) segment were each measured (tension).
All animals ate a standard solid chow diet until 24 hours before operation, during which they were provided a liquid diet ad libitum (Bio-Serv, Frenchtown, NJ). Liquid diet was continued postoperatively until the animals could tolerate at least 100 mL per day and had passed stool, at which time standard solid chow was restarted.
2.1. Operative procedures 2.5. Histologic evaluation In all animals, a midline laparotomy was performed. Isolated, Roux-enY segments were created in each animal: the bowel was the eviscerated and a location approximately 30 cm from the ligament of Treitz (LOT) was selected for transection. An anastomosis was then created between the proximal cut end and a location 6–10 cm from the distal cut end [single layer, interrupted 6-0 PDS (Ethicon, Somerville, NJ)], leaving a length of isolated small intestine (the Roux limb). For C1 animals, the blind end of the Roux limb was ligated and the abdomen was closed with no further intervention. In both C2 and Exp animals, the blind end was wrapped around a coiled, cylindrical shape-memory polymer device. The blind end was then closed and sutured to the side of the roux limb (Figs. 1 and 2). In the C2 animals, this device did not expand, while in Exp animals, the device expanded radially (Fig. 2).
At necropsy, a segment of small bowel from the Roux limb (control 1) or wrapped segment (control 2, experimental) were preserved in 4% formalin. Care was taken to avoid areas adjacent to sites of anastomosis. They were then embedded for frozen section, sectioned, stained with hematoxylin and eosin, and reviewed by a gastrointestinal pathologist who was blinded to operative group. Mucosal thickness/crypt depth, thickness of internal muscularis propria (the circular layer), villous height and width were measured in each sample with cellSens digital imaging software (Olympus, USA) according to manufacturer's instruction. The parameters were evaluated in 10 distinct sites and the average was recorded. Crypt density and fission rate were determined using previously validated methodology [17].
2.2. Device 2.6. Total DNA and protein A sheet of SMP was rolled into a cylinder, which was designed to expand radially over 7 days when activated by body heat and moisture. To create the polymer, isobornyl acrylate, 2-hydroxyethylacrylate, and 1,6-hexanediol diacrylate were mixed in weight ratio of 75:20:5, respectively. 0.5 wt% of phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide was used as a photoinitiator. All reagents were used as received (Sigma-Aldrich, St. Louis, MO). The mixture was injected into a glass mold with a 0.86 mm spacer and photopolymerized for 3 minutes at 405 nm using a LED flood lamp. After mold removal, the polymer was cut with a CO2 laser (Gravograph LS-500, Gravotech, Inc, Duluth, GA) into 15 mm × 47 mm rectangles with rounded edges. In order to program the temporary shape, the thin rectangles were wrapped around a mandrel in 80 °C water to form a cylinder with a diameter of 8.5 mm and height of 15 mm. They were constrained with plastic ties while cooling to store the temporary shape.
Measurements of total DNA and protein contents in the isolated bowel segment wall were performed in snap-frozen fresh samples based on methods as previously described [18]. Briefly, DNA and protein were isolated using the All Prep DNA/RNA/Protein Mini Kit (Qiagen, Gaithersburg, MD) per manufacturer's instructions. DNA was quantified using a NanoDrop 8000 (Thermo Scientific, Waltham, MA), with nucleic acid concentrations reported as mg/mL. Total protein was determined using the colorimetric DC Protein Assay (BioRad, Hercules, CA) per manufacturer's instructions, based on the reaction of protein with an alkaline copper tartrate solution and Folin reagent. Absorbances were read at 750 nm on a microplate reader (FLUOStar Omega; BMG Labtech, Cary, NC). A standard curve was obtained using known concentrations of bovine serum albumin provided by the manufacturer. Protein concentrations were also reported as mg/mL.
Fig. 1. Schematic for experimental animals; creation of Roux-en-Y limb, placement of cylindrical coil and radial expansion (over 7 days).
Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
J.G. Fisher et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
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Fig. 2. Photographs at operation. Radial expanding shape memory polymer device (A) placed with end of Roux limb (B) wrapped around it (C and D). Control group 2 underwent the same operation with placement of a nonexpanding polymer coil.
2.7. Statistical analysis
4. Discussion
Animals surviving to 14 days were included. Analysis was performed using JMP Pro version 10.0 (SAS Institute, Cary, NC). Change in length, percent change in length, all histologic measures, and DNA:protein were all compared using Student's t-test. All values are presented as mean ± standard deviation.
Distraction enterogenesis, or creating intestinal tissue by mechanical stretch, is a promising new therapy for short bowel syndrome (SBS). The applied tension induces growth via several known molecular mechanotransduction pathways [19], stimulates mesenteric neovascularization [20], and stretched jejunal segments appear to function well when replaced into continuity [21]. Despite the number of devices now described in the literature, none has yet translated into clinical practice. The extant approaches are relatively complex and have significant limitations, such as the need to violate the bowel wall, the use of a closed loop, or the need for a complex activating mechanism [7–12]. This investigation demonstrates that a self-expanding polymer placed in a simple extraluminal configuration can elongate small bowel and appears to induce growth. The percent change in length was significantly higher in the experimental group compared with the device control. Because only a short portion of the long Roux limb was stretched, the lack of difference in Roux length is not surprising (a ~2 cm portion of the ~10 cm limb was stretched by an additional ~2 cm, a large change relative to the experimental segment but a small change relative to the whole limb). The extraluminal approach used here doubled the length of the target segment on average, which is a similar degree of increase to other techniques [7–12]. To produce this degree of elongation, the device only increased in diameter by 70%, substantially less than those used in other approaches. The intraluminal devices used previously produce tension by expanding longitudinally along the axis of the bowel. This configuration requires that the device expand as much or more than the intestine is expected to stretch (~ 1:1 ratio). By wrapping the bowel around a radially-expanding device, it stretches with the circumference of the implant, meaning that each increase of 1 mm in device diameter produces 3.14 (or 1 × π) mm in bowel stretch. Thus this design potentially allows for a greater increase in bowel length for each increment of device expansion. A more compact device will be advantageous in patients where a substantial degree of growth is needed, but the dimensions of the abdominal cavity limit the maximum implant size. It is possible that in the future this advantage may be further multiplied by wrapping multiple loops around the device. The lack of difference between groups in both muscularis thickness and DNA:protein ratio suggests that the elongation seen is reflective of actual tissue growth rather than simple stretch with thinning of tissue. Though evaluating markers of cellular proliferation potentially may have enhanced such an analysis, this approach may have been confounded in our model by the presence of fibrotic tissue that developed in proximity to the device. Future studies will utilize techniques that can appropriately delineate histologic types and measure proliferation in intestinal tissue [22]. No differences were seen among histologic markers for intestinal adaptation such as villus height, crypt depth, and crypt fission rate. However, the distal portion of the Roux limb that was stretched was remote from the flow of luminal nutrients, which is an important stimulus for typical mucosal adaptation [23],
3. Results The overall survival rate was 73% (19/26). Deaths occurred in each group and were attributed to stricture or leak at the Roux-en-Y anastomosis or complications of anesthesia. There were 5 animals surviving to 14 days in the C1 group, 6 in C2, and 8 in Exp. Weight gain was similar between groups (P = 0.68). Bowel measurements at operation (day 0) and necropsy (day 14) are shown in Table 1. The Roux limb length of the two control groups C1 and C2 were measured in situ (0 ± 0% vs 20 ± 46%, P = 0.06) and under standard tension (0 ± 21% vs 3 ± 34%, p = 0.37) at necropsy, demonstrating no change in length. SMP devices in the Exp group increased from 28 ± 2 to 47 ± 10 mm in circumference (70% increase). Experimental segments increased from 24 ± 3 mm to 50 ± 15 mm in the Exp group and 35 ± 9 mm to 37 ± 6 mm in the C2 group when measured on standard tension (percent change 107 ± 52% vs 7 ± 31%, P = 0.001). There was a variable amount of fibrosis around the device and experimental segment in both Exp and C2 groups. These adhesions were removed after in situ measurements were made. There were no significant differences in villus height, width, muscularis thickness, crypt density, or crypt fission rate between the three groups (Fig. 3). Crypts were less deep in the C1 group than C2 or Exp (P = 0.02). The total DNA masses in each group were: C1 0.015 ± 0.017, C2 0.008 ± 0.008, Exp 0.006 ± 0.003 mg. Total protein masses were: C1 0.33 ± 0.05, C2 0.27 ± 0.03, Exp 0.29 ± 0.06 mg. There were no differences in DNA:protein between groups (Fig. 4, P = 0.41).
Table 1 Lengths of wrapped bowel segments at operation and necropsy (day 14). Measurements were made on the antimesenteric border in situ and under standard (6 g) tension ex vivo (at necropsy). Wrapped Segment Length: Control (C2) Change (mm) In situ Tension Percent change In situ Tension
Experimental
p
11 ± 7 1 ± 10
28 ± 10 26 ± 13
0.003⁎ 0.002⁎
29 ± 20 7 ± 31
114 ± 31 107 ± 52
b0.001⁎ 0.001⁎
⁎ P b 0.05, Student's t-test.
Please cite this article as: Fisher JG, et al, Extraluminal distraction enterogenesis using shape-memory polymer, J Pediatr Surg (2015), http:// dx.doi.org/10.1016/j.jpedsurg.2015.03.013
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J.G. Fisher et al. / Journal of Pediatric Surgery xxx (2015) xxx–xxx
Fig. 3. Histologic measures. There were no differences between groups except in crypt depth: the crypts in the Control 1 group were less deep than the other two groups (*P = 0.02). All comparisons by ANOVA.
and thus a lack of difference may not be surprising. Further study using this technique to stretch bowel that remains in continuity is needed to clarify the effect of stretch on mucosal adaptation. This model has a number of advantages over previously studied approaches. Shape-memory polymers can expand when activated by body heat and water uptake and will expand at a relatively constant rate determined by the polymer's chemical design. This obviates the need for external activation and thus additional hardware. Compared with an expander that is incrementally enlarged, the consistent tension applied here creates a constant stimulus for growth and hence may be less likely to tear tissue. This device is also easily scalable to a large animal model for eventual human use. The authors anticipate that a loop of small intestine in continuity could be wrapped around a similar radially expanding cylindrical device and thus a potential future advantage is that this operative approach would not require extensive dissection, violation of the intestinal wall, or the creation of a closed loop, which are likely to cause pain and potentially other complications. Further, though none of the devices used to stretch bowel are FDA approved, an orthopedic device based on a methacrylate SMP has been FDA cleared [24] and the acrylic SMP used here has a chemistry similar to other plastics used in medical applications [25,26]. The small number of animals studied and the inherent variability in measuring intestinal length limit this investigation. Further, the devices expanded somewhat less than expected and thus less of an increase in length was found than initially planned. By altering the mechanochemical properties of the polymer, however, the force applied can be increased to allow for full deployment and preliminary results with updated polymers are promising. The variable fibrosis around the devices is incompletely explained by this study and may have limited expansion to some degree. Though the potential for serosal tears exists, none of the animals were found to have died of a perforation at the wrap site. Lastly, for this initial investigation, the stretched segments were not replaced
into continuity to test their function. The authors anticipate that this technology can be easily applied to a large animal model in which the function of the resulting stretched bowel could be rigorously examined. This initial series demonstrates the feasibility of an extraluminal model for distraction enterogenesis using a shape-memory polymer device that is easily installed, self-expanding, and scalable. It successfully produced elongation of intestine that appears attributable to growth. Extraluminal, radial expansion has the potential to provide more bowel stretch in a smaller space without the need to violate the intestinal wall or expose the mucosa to a foreign body. Further study to optimize this technique for potential human use is warranted. Acknowledgments The authors would like to hank Azra Ahmed, BS for technical assistance. Appendix A. Discussion Presented by Jeremy Fisher, Boston, MA Discussant: DR. JOSH HORVATH, Los Angeles, CA I'm wondering in your self-expanding polymer, how long does it take to deploy when exposed to body heat and how do you prevent breaking the suture line open? Response: DR. FISHER The nice thing about these polymers is that you can set them by use of the chemistry. You can determine exactly how long you want them to take to resume their initial confirmation. We program these to change back to their initial shape in a week. How do you know it's not going to hurt your suture line? I think that's a little bit of trial and error. That's certainly something we were worried about here. But in this group, the tension is very low, and we did not actually see disruption of the suture line at all. I think that's one of the things we can work out as we expand to a large animal model, but it seems promising in this experiment.
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Fig. 4. Mean DNA to protein ratio by group. Error bars represent standard deviation. P = 0.41 (ANOVA).
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