Bioadhesive polymers can serve as surgical sealants with a wide range of potential clinical applications, including augmentation of wound closure and acute induction of hemostasis. Key determinants of sealant efficacy include the strength and duration of tissue-material adhesion, as well as material biocompatibility. Canonical bioadhesive materials, however, are limited by a tradeoff among performance criteria that is largely governed by the efficiency of tissue-material interactions. In general, increasingly bioreactive materials are endowed with greater bioadhesive potential and protracted residence time, but incite more tissue damage and localized inflammation. One emergent strategy to improve sealant clinical performance is application-specific material design, with the goal of leveraging both local soft tissue surface chemistry and environmental factors to promote adhesive tissue-material interactions. We hypothesize that copolymer systems with equivalent bioreactive group densities (isoreactive) but different amounts/oxidation states of constituent polymers will exhibit differential interactions across soft tissue types. We synthesized an isoreactive family of aldehyde-mediated co-polymers, and subjected these materials to physical (gelation time), mechanical (bulk modulus and adhesion strength), and biological (in-vitro cytotoxicity and in-vivo biocompatibility) assays indicative of sealant performance. Results show that while bioadhesion to a range of soft tissue surfaces (porcine aortic adventitia, renal artery adventitia, renal cortex, and pericardium) varies with isoreactive manipulation, general indicators of material biocompatibility remain constant. Together these findings suggest that isore-active tuning of polymeric systems is a promising strategy to circumvent current challenges in surgical sealant applications.
Bioadhesive polymeric materials have an established history of medical use, with utilities ranging from acute induction of hemostasis in cases of trauma to augmentation of wound closure in surgical applications. [
Bioadhesive materials can be loosely divided into two categories which exemplify the current state of sealant technologies. On the one hand, there are numerous synthetic materials which adhere vigorously to the full range of soft tissues and persist at the site of application for long periods of time. Many of these materials are based on cyanoacrylate and its derivatives, wherein adhesive bonds with soft tissues are rapidly formed in the presence of trace water. [
To address the long-standing challenges limiting sealant use and efficacy, recent efforts have focused on tissue-specific material design. [
Classes of Sealants (Base Materials) | Potential Applications | Strengths | Weaknesses | Ref |
---|---|---|---|---|
SYNTHETIC MATERIALS | ||||
Cyanoacrylate | Dermal applications; Wound closure; Hernia repair | Rapid polymerization; high adhesion strength | High toxicity of degradation by-products | 23 - 25 |
Polyurethane | Orthopedic and renal surgery; Pancreatic occlusion; Vascular surgery | High elasticity; Moderate-high adhesion strength | Moderate toxicity of degradation by-products | 26 - 29 |
Poly(ethylene glycol) | Cranial surgery; Spinal surgery; Retinal applications | Moderate adhesion strength; High biocompatibility; Soft tissue-like mechanical properties | Significant/uncontrolled swelling | 30 - 32 |
NATURAL MATERIALS | ||||
Fibrin | Hemorrhage control; Wound closure | High biocompatibility; High hemostatic potential; Rapid curing in-situ | Low adhesion strength; High cost; Risk of disease transmission | 33 - 36 |
Albumin/glutaraldehyde | Vascular surgery; Cardiac surgery; Lung surgery | Moderate adhesion strength; Rapid cross-linking | Toxicity of cross-linking agent; Moderate biocompatibility | 37 - 39 |
Collagen/Gelatin | Lung surgery; Vascular surgery; Gastrointestinal surgery | Low risk of disease transmission; Low cost; Moderate-high adhesion strength | Toxicity of crossing-linking agent; Moderate biocompatibility (depending on formulation) | 40 - 42 |
Polysaccharides (including dextran and chitosan) | Lung surgery; Hemorrhage control | Tunable polymer microstructure; High biocompatibility; Hemostatic potential | Moderate adhesion strength | 15;19 - 20; 43 - 44 |
signing soft tissue sealants for universal deployment is fading, whereas tissue- and application-specific approaches are gaining momentum.
It is well-established that increasing polymer reactive group content will promote bioadhesion, albeit to different degrees and saturation levels when applied to various soft tissue surfaces. [
We synthesized a family of two-component, aldehyde-mediated bioadhesive materials composed of dextran aldehyde and chitosan polymers. In this experimental material system, both cohesive cross-linking within the material and adhesive cross-linking to local tissue surfaces are achieved through aldehyde-me- diated imine bond formation. Within our series of experimental materials, the dextran oxidation state and solid content are simultaneously varied such that total aldehyde concentration is fixed, i.e. this is a family of isoreactive material formulations. We assess key sealant properties and biological response variables following application of these materials to multiple soft tissue surfaces, and evaluate the potential for isoreactive tuning of bioadhesive materials to enhance tissue-specific interactions
All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the University of South Carolina’s Institutional Animal Care and Use Committee.
Dextran oxidation The synthesis of dextran aldehyde has been previously described. [
Aldehyde content Aldehyde content was determined via a previously described titration method. [
Chitosan synthesis A 2 wt.% chitosan solution (average molecular weight of 340 kDa, Sigma Aldrich) and 1% acetic acid solution were prepared and mixed at room temperature for 5 hours. The mixing period yielded a viscous homogenous solution, which was subsequently degassed and stored at room temperature until use.
Isoreactive co-polymers Four batches of dextran aldehyde with a range of percent oxidations were selected for subsequent studies. Given the aldehyde content of each batch (determined above), the wt.% of polymer required to form aqueous solutions with equivalent total aldehyde group content (isoreactive) was calculated. Four isoreactive dextran aldehyde solutions (A-D) were then prepared via completely dissolving the appropriate amount of oxidized dextran in DI water.
Co-polymer material systems were formed with dextran aldehyde solutions (A-D) in combination with the prepared chitosan solution. In all co-polymer systems, the aldehyde group density (of the dextran component) was 3-fold higher than the amine group density (of the chitosan component). To facilitate co-polymer cross-linking, dextran aldehyde and chitosan solutions were loaded into a dual-chamber syringe equipped with a 12-step mixing tip. Upon injection and controlled mixing, constituent polymers react via imine bond formation to yield a solid co-polymer (
Gelation time The gelation time of each co-polymer formulation is defined as the time required for solid globule formation following a 100 μL injection onto a glass surface maintained at 37˚C. The injected material was continuously agitated with a magnetic stirring rod, and solid globule formation was visually determined.
Compressive modulus Cylindrical test samples (diameter = 9.5 mm and height = 6.25 mm) were prepared via co-polymer injection into a silicon mold. Samples were allowed five minutes to cross-link, after which they were carefully removed from the mold. A uniaxial mechanical testing system (Bose® Biodynamic Test Instrument, Minnetonka, MN) configured for unconfined compression testing was used to apply a ramped displacement (5 mm total displacement; displacement rate of 0.005 mm/sec) to each sample. Force and displacement data were continuously recorded (data acquisition rate of 20 points/sec) using an integrated system software (Wintest®, Minnetonka, MN). The mechanical behavior of these materials was assumed to be linear, elastic, homogeneous, and isotropic,
and the materials were modeled as incompressible solids due to the high water content. In the context of these assumptions, recorded mechanical data were processed to yield true stress versus strain curves and ultimately calculate the compressive elastic modulus (E) of each test sample (i.e. slope of the stress-strain curve).
The morphology and mechanical strength of tissue-material interfaces formed between isoreactive co-polymer formulations (A-D) and select porcine soft tissue surfaces (aortic adventitia, renal artery adventitia, renal cortex, and pericardium) were quantified to reflect adhesive material properties. For the following ex-vivo studies, soft tissues were harvested from 7 - 12 month old swine and completed protocols within 2 hours of animal sacrifice.
Interfacial morphology To facilitate visualization of the tissue-material interface, fluorescently labeled co-polymer formulations were prepared via 0.5 wt % inclusion of fluorescence (6-fluorescein-5-carboxyamido hexanoic acid, Invitrogen) into the chitosan component as previously described. [
Adhesion strength Co-polymer adhesive mechanics were quantified using a previously described testing methodology. [
The biocompatibility of isoreactive co-polymer formulations (A-D) was assessed via in-vitro cytotoxicity studies and in-vivo sub-cutaneous implantation studies. While neither method is directly relevant to specific sealant applications, these studies provide general indications as to whether isoreactive design manipulations within the co-polymer system will likely impact material biocompatibility.
In-vitro cytotoxicity Primary rat fibroblasts (~7e4 cells/mL) were seeded on to 24 well plates and cultured to confluence using standard media (Cell Applications, Inc.). Each well plate was then drained of media to facilitate direct injection of co-polymers (100 μL) onto the cell monolayer. Materials were allowed five minutes for cross-linking, after which fresh culture media was replenished within each well plate. Following a 48 hour incubation period, a neutral red uptake (NRU) assay (Sigma Aldrich) for cell viability/cytotoxicity was performed. The assay consist of a two hour co-incubation of cells/materials with the supravital dye (neutral red), a washing treatment, and subsequent quantification of absorbance. Obtained absorbance measurements were normalized with respect to control wells (identical cell cultures with no material exposure) and reported for each co-polymer formulation (A-D).
In-vivo studies Sterile sample preparations of co-polymer formulations A-D were prepared for subcutaneous implantation in adult male Sprague Dawley rats (180 - 220 g, Charles River Labs). A randomized pattern of five discrete subcutaneous dorsal implantation sites was assigned to each rat (n = 12). Each of four implantation sites was assigned one co-polymer formulation (A-D), wherein a 100 μL injection was sterilely delivered. The fifth implantation site was used for a sham procedure (100 μL saline injection). After 7 days, the rats were sacrificed and tissue was harvested for histological and molecular assays. For histological studies, tissue samples were fixed in 4% formalin, sectioned (20 μm thickness), and stained with hematoxylin and eosin (H & E). Histological images (40X) were subjected to blind scoring, where the inflammatory cells present in four randomly selected regions (25 mm2 regions, total area of 100 mm2 per slide) were counted and summed. Additional tissue samples collected from each implant site (n = 3 per material & sham group) were snap-frozen upon acquisition and later used to quantify local interleukin (IL) levels. Tissue samples were thawed, homogenized, and analyzed using the Bio-Plex Pro Assays Quick Guide (Bio- Rad), enabling quantification of local IL-1β, IL-2, and IL-6 concentrations (assay sensitivity of 0.8 - 2.0 pg/mL).
Obtained data were analyzed using Mann?Whitney tests for significance between groups and Wilcoxon rank tests for pair-wise comparisons within groups, with groups defined by co-polymer formulation (experimental groups) or included as controls (sham procedure for in-vivo studies). Differences were considered to be significant if p-value < 0.05.
Isoreactive material synthesis yielded four dextran aldehyde-chitosan co-poly- mer formulations (A-D) that facilitate investigation of the proposed material design strategy (
Assays were conducted to determine if key intrinsic properties for surgical sealant applications vary in response to isoreactive manipulation. Specifically, the impact on co-polymer gelation kinetics (mean time for liquid-solid phase transition under controlled component mixing conditions) and the compressive elastic modulus (determined via unconfined uniaxial compression testing of cylindrical material samples) were determined. Among the examined co-polymer formulations, no significant differences were found in either mean gelation times or compressive moduli (
Co-Polymer Formulation | Dextran Aldehyde | Chitosan | Co-Polymer | |||||
---|---|---|---|---|---|---|---|---|
Molecular Weight (kDa) | Percent Oxidation (%) | Solid Content (%) | Aldehyde Content (#per mL) | Molecular Weight (kDa) | Solid Content (%) | Amine Content(#per mL) | Reactive Group Ratio (CHO:NH2) | |
A | 40 | 24.3 | 10.8 | 2.30 × 1020 | 340 | 2 | 7.63*1019 | 3 |
B | 40 | 42.1 | 6.32 | 2.30 × 1020 | 340 | 2 | 7.63 × 1019 | 3 |
C | 40 | 53.5 | 4.93 | 2.30 × 1020 | 340 | 2 | 7.63 × 1019 | 3 |
D | 40 | 71.3 | 3.63 | 2.30 × 1020 | 340 | 2 | 7.63 × 1019 | 3 |
The adhesive interactions between co-polymer formulations and a range of soft tissue surfaces were assessed in terms of tissue-material interfacial continuity and maximal adhesion strength. The soft tissue surfaces considered were the aortic adventitia, renal artery adventitia, renal cortex, and pericardium, all of which are potential targets for clinical sealant applications (
Similar tissue-specific responses were found when adhesion was assessed from a mechanical perspective (
Assays to determine the cytotoxic effects of formulations A-D demonstrated that co-polymer formulations are similarly tolerated by the cell culture monolayer. All formulations maintained greater than 58% viability of the control wells, with no significant differences in cytotoxicity among the material formulations (
While cytotoxicity assays suggest reasonable and consistent material biocompatibility, complementary subcutaneous implantation studies were undertaken to quantify and compare the in-vivo tissue response to isoreactive co-polymers. Obtained results demonstrate no significant elevations in inflammatory cell count relative to sham, and no dependence on dextran oxidation state/solid content was observed among the formulations tested (
There are several study limitations that should be considered upon interpretation of our findings. First, we have not directly shown that the surface-present biochemical groups (amine groups) targeted for adhesive bond formation in fact have different densities/spatial distributions among tissue surfaces. While beyond the scope of the present study, the tissue-present amine group distribution could be quantified with the use of functional atomic force microscopy (fAFM). [
relevant (reciprocal) reactive groups would provide a means to directly test the proposed approach to enhance bioadhesion. Second, while bioadhesion strength and interfacial morphology were assessed in a tissue-specific manner, we only provide general measures of material biocompatibility (in-vitro cytotoxicity and in-vivo tissue response following subcutaneous implantation). Moreover, the time point of the in-vivo studies (7 days post implantation) may have failed to detect the peak of the inflammatory response, which would likely occur upon material erosion and by-product generation. More comprehensive evaluation of the proposed design strategy, specifically the insensitivity of biocompatibility to isoreactive design manipulation, requires assessment of local tissue response in various implant scenarios and over the complete residence time of the material.
The aim of this study was to evaluate a novel approach for tissue-specific design of surgical sealants. Specifically, we investigated the potential for isoreactive tuning of polymer design variables to enhance tissue-material adhesion without compromising biocompatibility. Using an experimental aldehyde-mediated co- polymer system, we were able to demonstrate that for select tissue types, isoreactive titration of constituent polymer oxidation state and solid content impacts bioadhesion in a tissue-specific manner, and conversely do not impact generalized indicators of material biocompatibility. These findings imply that for a given clinical application (targeted tissue type), isoreactive tuning of a surgical sealant can be optimized such that adhesion is maximized while material biocompatibility remains at a baseline level that is determined by other factors (most notably the overall bioreactive group content of the material). Although only demonstrated in our experimental material system and with a limited number of soft tissue types, we expect that this design concept can be extended to a broad range of bioadhesive materials that target a specific surface-present chemical group for adhesive bond formation.
All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the University of South Carolina’s Institutional Animal Care and Use Committee.
Competing InterestsThe authors declare that they have no competing interests
FundingThis research was supported by the NIH INBRE Grant for South Carolina (P20GM103499, to TS and MJU) and a VA Merit award (BX000168-06, to FGS).
Authors’ ContributionsMJU, FGS, and TS conceived and designed all studies. JF, ER, AM synthesized and characterized all co-polymers. AM, HD, FGS performed and analyzed implantation studies. JF, MJU, FGS, and TS prepared the manuscript. All authors read and approved the final manuscript.
Ferdous, J., Romito, E., Doviak, H., Moreira, A., Uline, M.J., Spinale, F.G. and Shazly, T. (2017) Isoreactive Manipulation of Bioadhesive Polymers Impacts Tissue-Specific Interactions. J. Biomedical Science and Engineering, 10, 287- 303. https://doi.org/10.4236/jbise.2017.105022