Author + information
- Received October 17, 2005
- Revision received February 9, 2006
- Accepted February 14, 2006
- Published online July 4, 2006.
- Christian Jux, MD⁎,⁎ (, )
- Harald Bertram, MD†,
- Peter Wohlsein, DVM‡,
- Michael Bruegmann, DVM‡ and
- Thomas Paul, MD, FACC⁎
- ↵⁎Reprint requests and correspondence:
Dr. Christian Jux, Department of Pediatric Cardiology and Pediatric Intensive Care Medicine, Georg-August University, Robert Koch Strasse 40, D-37099 Goettingen, Germany.
Objectives We sought to test the hypothesis that interventional atrial septal defect (ASD) closure can be performed safely and effectively using a bioresorbable occluder matrix.
Background The ideal septal occluder scaffold should promote the healthiest and most complete healing response while eventually facilitating the full resorption of the material and leaving “native” tissue behind, thus minimizing the potential for future complications from chronic foreign body and maintaining the possibility for later unobstructed transseptal access to the left atrium.
Methods The STARFlex occluders (NMT Medical Inc., Boston, Massachusetts) were modified by substituting the conventional polyester fabric for a bioengineered, acellular type-I collagen matrix derived from porcine submucosa with a heparin-coated surface (BioSTAR occluder, NMT Medical Inc.). Comparative transcatheter closure of ASDs was performed in young sheep (n = 36). Gross pathology and histopathology were obtained after follow-up periods ranging from 7 days to 2 years.
Results The STARFlex (control) devices were encapsulated time-dependently by ingrown fibrous tissue. Histology showed a mild but chronically persisting foreign body reaction. By contrast, BioSTAR devices exhibited a mild-to-moderate transient cellular immune response. Heparin coating of the BioSTAR surface improved the biocompatibility of the device by reducing surface thrombogencity. A remodeling process of the collagen scaffold, starting after 30 days in vivo, resulted in the full replacement of the matrix by host tissue after 2 years of follow-up.
Conclusions The BioSTAR device is the first septal occluder with a totally bioresorbable matrix that is fully replaced by host tissue during the healing process. The promising results of this study support testing of the BioSTAR device in clinical trials.
Transcatheter closure of atrial septal defects (ASDs) has become a routine treatment modality for children as well as older individuals. The permanent implant is expected to persist for the natural life expectancy of some 70 or 80 years in the young recipients. However, the device itself may be dispensable once the defect is closed and covered by a complete and firm layer of ingrown, endogenous “repair” tissue. This thought led us to the idea of a biodegradable occluder matrix: an absorbable scaffold that acts as a temporary guide rail for the ingrowing host tissue from the defect edge to cover the device and thus the defect underneath. The ideal bioresorbable septal occluder scaffold should promote the healthiest and most complete healing response possible while eventually facilitating the full resorption of the material, leaving “native” tissue behind and minimizing the potential for future complications while providing the possibility of later unobstructed transseptal access.
Despite considerable differences between the designs of the various septal occluder implants, all current commercially available devices consist of a metal framework and a synthetic fabric. Studies in experimental animals have characterized the histologic features and time course of the healing response and tissue ingrowth onto the device after implantation (1–7).
Although potential short- and mid-term complications such as residual shunts, thromboembolism, friction lesions, perforations, metal fractures, and rhythm disturbances have been well described (8–11), the long-term potential for adverse effects over several decades remains to be determined. At the same time, technologies are just now evolving for the treatment of left-sided heart disease, including percutaneous heart valve repair or replacement, arrhythmia studies, and therapies (e.g., pulmonary vein exclusion and left atrial appendage closure). An increasing proportion of the young population is likely to benefit from these technologies in the future. The maintenance of an unobstructed trans-septal access to the left atrium is of paramount importance for these interventional procedures, which may be hampered by metal-rich devices and synthetic fabrics.
The aim of this preclinical feasibility study was to provide a proof of the concept of biodegradable interventional ASD closure devices and to assess the efficacy and safety of a degradable occluder matrix in an experimental animal model.
ASD occluder devices
CardioSEAL STARFlex devices (NMT Medical Inc., Boston, Massachusetts), consisting of a MP35N stainless steel framework and a knitted polyester fabric, served as control devices (Fig. 1A).In BioSTAR devices (NMT Medical Inc.) with bioresorbable fabric, the polyester was substituted by a bioengineered, highly purified, acellular matrix consisting primarily of type I collagen (Organogenesis Inc., Canton, Massachusetts). Figure 1B shows the BioSTAR device after rehydration of the matrix with saline. This biological matrix was derived from porcine submucosa. Briefly, porcine small intestine is mechanically stripped to obtain the tunica submucosa and remove the tunica mucosa as well as tunica muscularis and serosa. Non-collagenous components such as cells, cellular debris, nucleic acids, lipids, and other extracellular matrix proteins such as glycosaminoglycans and proteoglycans are removed by treatment of the tissue with alkali, chelating agents, acids, and salts. This detergent- and enzyme-free method preserves the structural integrity, cell compatibility, strength, and bioremodelability of the collagen matrix (12). The purified intestinal collagen layer (ICL) is then reinforced by bonding several collagen layers together using a thermal bonding technique. The thickness of ICL used in this study was 150 to 200 μm. After the suture of the ICL patches onto the device framework, the fabric was heparin-coated by a heparin benzalkonium chloride dip. Two devices were left without a heparin coating. Devices were sterilized with gamma irradiation at 25 kGy.
Animal model/defect creation
Atrial septal defects were created in young female black-headed mutton sheep (n = 36, mean body weight 32 ± 5 kg) by trans-septal puncture and subsequent balloon dilation of the interatrial septum (1,13) under fluoroscopy and intracardiac echocardiography (ICE) control using the AcuNav system (Acuson, Siemens Inc., Mountain View, California). Anesthesia was introduced by xylazine 0.4 mg/kg intramuscularly and an intravenous bolus infusion of 10 mg/kg ketamin. Animals underwent endotracheal intubation and were mechanically ventilated with isofluorane and oxygen/room air. Electrocardiogram, heart rate, respiratory rate, transcutaneous oxygen saturation, tidal volume, and end-tidal CO2were monitored throughout the procedure.
Following trans-septal puncture, a defect within the interatrial septum (Fig. 2A)was created by subsequent balloon dilation of the interatrial septum using balloons with diameters of 6, 10, and 15 mm, respectively (Opta Pro, Cordis Europe N.V., Roden, the Netherlands; and Balt CBV, Montmorency, France) over a guidewire inserted into the left atrium or a pulmonary vein. Heparin (200 U/kg after trans-septal puncture) and antibiotics (flucloxacillin 1.5 g at the start of the procedure) were administered intravenously.
All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85-23, revised 1996). The study was approved by the local governmental animal ethics committee.
Device implantation/defect closure
After allowing for healing of the created ASD edges for two to three weeks, animals were divided randomly into two groups. Defect closure was performed using either conventional 23-mm STARFlex devices (control group, total n = 10) or 23-mm BioSTAR devices (total n = 26), respectively. Animals were anesthetized as described earlier. Before defect occlusion, heparin (200 units/kg intravenously) and antibiotics (flucloxacillin 1.5 g intravenously) were administered and balloon sizing of the defect was performed (Arrow, Reading, Pennsylvania) by ICE using color Doppler to assess for stop of flow. All devices were deployed transvenously through a 10-F–long Mullins-type transseptal sheath (Cook Inc., Bjaeverskov, Denmark) under fluoroscopy and ICE control as described previously (14). Color Doppler was used to assess potential residual shunting. Animals did not receive any anticoagulation during follow-up.
Device explantation/macroscopic and histologic examination
One day before sacrifice of animals with more than seven days of follow-up, device position was confirmed by both fluoroscopy and ICE control under general anesthesia as described earlier. A color Doppler examination was performed to assess residual shunting. The degree of residual shunts was quantified according to the protocol published by Boutin et al. (15). Animals from both groups were sacrificed 7 days, 30 days, 90 days, 180 days, 1 year, and 2 years, respectively, after defect closure (2 STARFlex control devices and 4 BioSTAR devices per time point). The 2-year follow-up group consisted of 4 BioSTAR devices only; in the 7-day group 2 BioSTAR devices without heparin coating were added.
Before sacrifice, each animal received an intravenous dose of heparin (400 U/kg body weight) to prevent postmortem clot formation on the device, followed by a lethal injection of pentobarbital. Complete autopsy was performed in each of the animals by two veterinary pathologists. The hearts and adjacent vessels were inspected for the gross appearance of the implanted device and then fixed in alcoholic formaldehyde (two parts ethanol 95%, one part formaldehyde [40% gas by weight] [vol/vol]) for 48 h. In all animals, sections from 6 to 8 representative locations from the right and left atrial wall including the device (3 or 4 from the distal and 3 or 4 from the proximal umbrella, respectively) and from both ventricular and septal myocardium were taken. Tissue samples were embedded in paraffin wax, serially sectioned, and stained with hematoxylin and eosin (general stain and evaluation of cellularity) or elastica van Gieson (elastic fibers). All gross and microscopic specimens were reviewed by two experienced veterinary pathologists (P. W. and M. B.).
A two-tailed unpaired Student ttest was used for comparison of the means of defect sizes and device size to defect size ratios between the two device groups. A value of p < 0.05 was considered statistically significant.
All animals survived defect creation and the defect closure procedures as well as follow-up periods uneventfully. Interventionally created ASDs measured between 6 and 13 mm by two-dimensional echo (without stretching) and between 11 and 14 mm in diameter as determined by balloon sizing immediately before defect closure. The device-to-defect ratio thus varied between 1.6 and 2.1 with no statistically significant difference between the two groups. The STARFlex and BioSTAR devices were successfully implanted in all sheep. The technical handling of the BioSTAR devices did not differ significantly from that of the control devices. Likewise, no difference was experienced in the behavior of the BioSTAR device during loading, delivery, (re)positioning, or release of the device. Echogenicity of the collagen matrix as assessed by ICE was comparable to that of the polyester fabric, allowing for clear visualization of the device during deployment (Fig. 2B).
In the 30-day follow-up groups, one of the four BioSTAR devices and both STARFlex devices showed trivial residual shunts as assessed by color flow Doppler using ICE. In the 90-day follow-up groups, none of the four BioSTAR devices (but one of the two STARFlex devices) showed a trivial residual shunt. No residual shunting was observed in the 180-day and 1- and 2-year follow-up groups. All except one of the residual shunts (in a 30-day STARFlex explant) were confirmed later at postmortem examination. In all animals with residual leakage, a probe patent shunt was found at the margin of the device where the fabric was not firmly attached to the septum, resulting in space between fabric and septal wall.
Effect of heparin coating
Early thrombogenicity of the surface of the devices was compared after seven days in vivo. Heparin-coated BioSTAR devices showed considerably less deposition of blood-derived material on their surfaces than STARFlex control devices and uncoated BioSTAR devices (Figs. 3Ato 3F). Histologic workup of these devices confirmed the presence of less friable material consisting of deposited plasma proteins and blood cells on heparin coated BioSTAR devices (Figs. 3G to 3I). Thus, the defects covered by these devices could still be seen owing to the transparency of the matrix (Figs. 3C and 3F). Very early signs of a beginning neo-endothelialization were observed in the form of a cellular monolayer on the surface of these devices (Fig. 3I).
On gross inspection all devices were macroscopically intact. There were no metal arm fractures and no adverse effects from the centering spring. No significant compromise of blood flow at the pulmonary or systemic veins or at the heart valves was observed by before-sacrifice ICE, fluoroscopy, or postmortem inspection in any of the animals.
Conventional STARFlex devices were almost completely covered, except for the central pin and the metal arms, by a shiny, glistening surface layer through which the polyester fabric could be seen after 30 days in vivo (Fig. 4A).Devices with a longer follow-up in vivo showed a small amount of whitish, dull tissue underneath a complete glistening surface layer. Although the thickness of the tissue layer between surface and polyester fabric increased somewhat after 180 and 360 days compared with 90 days in vivo, the fabric remained visible through this tissue. The metal framework of the device was covered only by a shining film after 90 days. However, after 180 days in vivo, the ingrown tissue covered the straight parts of the metal arms, leaving merely the central pin and shoulder and elbow joint coils unincorporated (Figs. 4E and 4G).
Occasionally an infolding (“scalloping”) of the edges of the polyester fabric was observed, which prevented a smooth continuity between the devices and the adjacent atrial tissue. None of the devices showed any evidence of thrombus formation.
At gross pathology, BioSTAR devices showed a complete shining, glistening coverage on the device after 30 days in vivo (Fig. 4B). Compared to STARFlex control devices, BioSTAR occluders showed a significantly more thorough coverage of the device by grayish-white tissue at any time of follow-up during the entire study (Fig. 4).
Infolding (“scalloping”) of the edges was not observed in the BioSTAR explants. Rather, in comparison to control devices, the BioSTAR devices exhibited a smoother interface toward the adjacent atrial tissue. However, in some occluders there was a tendency for contraction in the middle of the edges of the ICL matrix. Again, none of the devices showed any evidence of thrombus formation.
Histology of healing response and bioresorption of the occluder matrix
The histologic examination of STARFlex devices after 30 days in vivo revealed a low amount of loosely arranged and poorly vascularized young granulation tissue covered by neo-endothelium (Fig. 5A).At later stages during follow-up, the ingrown tissue on the polyester surface was found to be organized in a longitudinal orientation parallel to the surface of the device (Figs. 5B and 5C). After 180 days in vivo, encapsulation of the polyester was observed. The typical histologic finding consisted of a bilayered texture: dense avascular connective tissue was observed immediately underneath the neo-endothelium, whereas more loosely arranged fibers containing capillaries and small vessels were found in deeper segments of the ingrown tissue (Fig. 5C). The amount of newly formed collagen and elastic fibers increased over time.
Directly adjacent to the polyester, there was a mild infiltration with lymphocytic cells, which persisted during the later stages of follow-up. Consistently, macrophages and multinucleated foreign body giant cells were found neighboring the polyester fibers (Fig. 5D). Histologic workup of BioSTAR devices explanted after 30 days in vivo showed complete neo-endothelial coverage that was in smooth continuation with the atrial tissue surrounding the previous defect. Multiple layers of young granulation tissue were found between the surface and the collagen implant (Figs. 6Aand 6B). Although largely intact, focal areas of the BioSTAR matrix exhibited the first signs of disintegration. Longitudinal splits and fraying at the terminal edges of the collagen matrix allowed for infiltration of the matrix with fibroblastic and mononuclear lymphoid cells (Figs. 6A to 6D). Starting after one month in vivo, phagocytosis of the matrix by macrophages and multinucleated giant cells was observed in direct contact with the collagen (Fig. 6C). The phagocytic activity continued (Figs. 6F and 6J) until full resorption of the implanted matrix was achieved.
Three months after implantation, progressive stages of BioSTAR matrix disintegration were found. Thicker stripes of fibrous granulation tissue extended into the collagen fibers, pushing apart detached collagen fragments. In some sections, the matrix layer appeared discontinuous (Figs. 6E and 6F). Compared with earlier explants, the sub-neoendothelial fibrous tissue was thicker, arranged in a more compact formation, and less rich in fibroblastic cells. Small vessels or capillaries and newly formed collagen fibers were observed within deeper layers of this tissue.
At later stages during follow-up, after 6 to 12 months in vivo, the ICL matrix had lost its structural continuity (Figs. 6G and 6I), although with some inter- and intraindividual variability; many sections exhibited only collagen fragments. In some sections, focal lymphocytic infiltration was found in the vicinity of the matrix (Fig. 6H). However, these lymphocytic infiltrations were always circumscribed and not seen in any area where the collagen had undergone full degradation.
After two years in vivo, the implanted collagen matrix was completely resorbed and fully replaced by autologous host tissue in 63% of all specimen locations (i.e., all serially cut sections in these locations were free of previously implanted collagen). In the remaining locations, only a few sections showed small remnants or tiny spots of the previously implanted ICL material. On the basis of these findings, it was calculated that approximately 90% of all implanted ICL material had been resorbed after two years in vivo. In comparison to the histology of earlier stages, the autologous repair tissue appeared relatively poor in fibroblastic cells but richer in connective fiber content. Especially the amount of newly formed elastic fibers increased over time (elastica van Gieson staining) (Figs. 6K and 6L). However, no fibrous capsule formation was found.
Technical aspects of the BioSTAR device
The general handling of the BioSTAR device did not significantly differ from that of the STARFlex control device, although the operators described the BioSTAR device behavior as somewhat “softer” compared with the STARFlex. Intracardiac echocardiography was used to guide the interventional defect creation as well as defect closure because the sheep anatomy does not allow for adequate transesophageal imaging of the interatrial septum. The BioSTAR occluder matrix showed appropriate echogenicity, allowing for precise visualization by ICE during implantation and follow-up studies.
Healing response to control and BioSTAR devices
The healing response to STARFlex control devices described in this study was uncomplicated and resembled that described previously by others both in experimental animals (1,2) and humans (16). Complete neo-endothelialization in all sections was observed after three months in vivo. Ingrowth of granulation tissue started in the device periphery and proceeded preferably along the framework arms toward the central parts of the device. The thickness of the tissue layer on the device surface increased over time, finally reaching a steady state after one year where only some arm joints remained unburied by tissue. A mild to moderate but persistent foreign-body reaction was noted in our study as well as in previously reported studies close to the polyester fabric (2,16).
Intestinal collagen layer, which has been used as the occluder scaffold on BioSTAR devices, is a highly purified acellular bioengineered type I collagen derived from porcine submucosa. It is gradually resorbed by the host organism and subsequently replaced by host tissue (12,17). Following extensive experimental investigations, porcine small intestinal submucosa has been successfully used clinically in various fields of soft tissue augmentation, to treat hernias and in tendon repair (18–20), stress urinary incontinence (21), leg ulcers (22), plastic surgery (23), and vascular surgery (24).
The BioSTAR devices showed an accelerated healing response compared with the control implants. First signs of a single-layer neo-endothelium were observed already after 7 days in vivo (Fig. 3I). A state of complete neo-endothelialization was observed after 30 days (Figs. 6A and 6B). This early neo-endothelialization argues in favor of the biocompatibility of the BioSTAR device. A layer of ingrown young granulation tissue that was in smooth continuity with the adjacent atrial tissue was found on the BioSTAR devices. The BioSTAR device showed an excellent efficacy in ASD closure. Although animal numbers involved in this study are too low to reach statistical power, the observed lower incidence of early residual leakage with the BioSTAR device is potentially attributable to the better alignment of the device to the surface of the atrium. Furthermore, the fact that the BioSTAR matrix is a foil rather than a knitted mesh may also decrease early residual shunting as observed as “foaming” through the polyester. However, larger scale clinical studies will be needed to verify this hypothesis.
Thrombogenicity of control and BioSTAR devices
Contact between blood and a foreign material is known to activate the coagulation and complement systems. Thrombus formation and thromboembolism due to the thrombogenicity of the implanted occluder surface are rare but clinically well-described potential complications. The vast majority of thrombi formations on ASD occluder devices occur in the early vulnerable phase before a protective complete neo-endothelialization on the device surface has developed (8,25). The BioSTAR devices as used in this study were heparin-coated, which led to a reduced thrombogenicity of their surface. The amount of blood cells and deposited plasma protein on the surface after seven days in vivo was significantly lower compared with both the uncoated BioSTAR devices and the polyester matrix of conventional STARFlex devices. Heparin surface coatings have been shown to reduce blood activation upon contact with various artificial surfaces, such as bypass tubings, intravascular catheters, stents, and Nitinol devices (26–28). The surface-bound heparin triggers a cascade of effects, including the initial interaction between heparin and antithrombin, reducing fibronectin deposition, inhibition of complement and platelet activation, and reduction of an initial inflammatory response (29–31).
Immunogenicity of control and BioSTAR devices
The ICL collagen matrix exhibited a surprisingly low immune response, given its xenogenic origin. Whereas the polyester fabric showed a mild but chronically persisting foreign body reaction, the BioSTAR matrix led to a transient, focally circumscribed mild-to-moderate lymphocytic immune response. Cellular infiltration was not observed once the implanted collagen matrix had undergone full degradation. The low immunogenicity may be explained by various facts. First, type I collagen, as the most abundant protein component of the extracellular matrix, is a highly preserved protein in evolution (32,33). Second, the particular cleaning process of the porcine submucosa removes cells and cellular debris, to which most of the immunogenicity is attributed, without damaging the native collagen structure (12,34,35). Finally, molecular studies showed that porcine small intestinal submucosa elicits a predominantly TH2-restricted immune response, consistent with a bioresorbable remodeling reaction rather than a graft rejection (36).
Biodegradability of the BioSTAR matrix
The unique feature of the ICL matrix, however, is its biodegradability. Remodeling of the collagen matrix started after 30 days in vivo with host cell infiltration. Autologous fibroblasts were observed to infiltrate the collagen implant, leading to the deposition and rearrangement of new host collagen. After 180 days in vivo, a significant degree of degradation of the matrix was obvious. Resorption of the material occurred by phagocytosis. In addition, collagen is known to be metabolized by various endopeptidases such as matrix metalloproteinases, serine, cysteine, and aspartic proteases (37). Through its ability to interact with the host, the purified collagen first acts as a scaffold for new tissue ingrowth. The matrix assimilates with host tissue as it is initially augmented by host tissue before it is resorbed, finally leaving nothing but autologous tissue behind.
This study proves the feasibility of the concept of biodegradable interventional ASD closure devices. The BioSTAR device is the first septal occluder device with a totally biodegradable matrix. It is, therefore, a step away from a mere mechanical septal defect occluder toward a septal repair device. Although the BioSTAR device only leaves a minimal amount of foreign material (i.e. the framework) behind, thus minimizing the potential risk for future complications from chronic foreign body and facilitating future trans-septal access to the left atrium, work is currently underway to develop a resorbable and fully retrievable occluder framework. This research may eventually result in various totally bioresorbable cardiovascular devices.
Because of the promising results of this experimental study, the BEST (Biostar Evaluation STudy) trial, the first in human investigation of this new technology, has been scheduled.
The excellent technical help of M. Zutz, RN, during the animal experiments and B. Buck in histotechnological workup is gratefully acknowledged.
This study was supported in part by a grant from the Bundesministerium für Bildung und Forschung (German Federal Ministry of Education and Research, BMBF no. 03N4023 to Dr. Jux) and NMT Medical Inc., Boston, Massachusetts.
- Abbreviations and Acronyms
- atrial septal defect
- intracardiac echocardiography
- intestinal collagen layer
- Received October 17, 2005.
- Revision received February 9, 2006.
- Accepted February 14, 2006.
- American College of Cardiology Foundation
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