Biomacromolecules. 2017 Aug 14; 18(8): 2529–2538.
Published online 2017 Jul 12. doi: 10.1021/acs.biomac.7b00683
PMID: 28699748
This article has been cited by other articles in PMC.
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Abstract
In order to prevent hemorrhage duringsurgical procedures, a widerange of hemostatic agents have been developed. However, their efficacyis variable; hemostatic devices that use bioactive components to acceleratecoagulation are dependent on natural sources, which limits reproducibility.Hybrid devices in which chain-end reactive poly(ethylene glycol) isemployed as active component sometimes suffer from irregular cross-linkingand dissolution of the polar PEG when blood flow is substantial. Herein,we describe a synthetic, nonbioactive hemostatic product by coating N-hydroxysuccinimide ester (NHS)-functional poly(2-oxazoline)s(POx-NHS) onto gelatin patches, which acts by formation of covalentcross-links between polymer, host blood proteins, gelatin and tissueto seal the wound site and prevent hemorrhage during surgery. We studieddifferent process parameters (including polymer, carrier, and coatingtechnique) in direct comparison with clinical products (Hemopatchand Tachosil) to obtain deeper understanding of this class of hemostaticproducts. In this work, we successfully prove the hemostatic efficacyof POx-NHS as polymer powders and coated patches both in vitro andin vivo against Hemopatch and Tachosil, demonstrating that POx-NHSare excellent candidate polymers for the development of next generationhemostatic patches.
1. Introduction
Oneof the main challengesduring surgical procedures on parenchymatoustissue is to attain control over bleeding.1 Suture control, electrocautery,2 andultrasonic sealing3 often do not sufficeduring operations on for example liver or kidneys. As a result, procedureslike hepatic resections4 or partial nephrectomy5 require an alternative approach to control bleeding.For this purpose, a wide range of topical hemostatic products hasbeen developed and are clinically available.6−8
The mainclass of hemostatic products is composed of polymericmaterials of biological origin such as starch,9 chitosan,10 oxidized regenerated cellulose,11,12 collagen, and gelatin.13,14 These biocompatibleand biodegradable products accelerate the natural coagulation cascadeand are available in various forms (powder, sponges, dressings). Theirhemostatic action is generally limited, for example when large areasof profuse bleedings should be treated. Moreover, animal-derived productscarry the risk of transmission of diseases via viral or prion agents.Within this class, Tachosil (a collagen carrier coated with humanderived fibrinogen and thrombin) is considered a “gold standard”due to its widespread use in liver resection.15 However, the high costs and the use of human derived materials havestimulated the search for alternative synthetic hemostatic products.As a result, synthetic polymer sealants16 have been developed which act independently of the natural coagulationcascade by their ability to seal off the wound surface, thereby stoppingthe blood flow. Although the efficacy of these products is reportedto be superior over naturally derived hemostats, the unknown biodegradation/excretionprofile of some of these polymers as well as their toxicity (e.g.,for cyano-acrylates)17 are drawbacks ofthis class of materials.
A more recent approach entails thedevelopment of hybrid products,which combine the beneficial properties of both synthetic and naturalpolymers. Two examples are Veriset18 (anoxidized regenerated cellulose sheet impregnated with trilysine and N-hydroxysuccinimide ester functional 4-arm poly(ethyleneglycol) (PEG-4-arm NHS)) and Hemopatch19−21 (a porous collagen carriercoated with PEG-4-arm-NHS). Both products have shown improved hemostaticefficacy compared to carriers without this coating21,22 and other commercially available products.18,19 In case of Hemopatch, the mechanism of action is based on instantaneouscovalent cross-linking between PEG-4-arm-NHS and amines present intissues, blood proteins and the collagen carrier, which seals offthe wound site and allows firm fixation of the patch to the tissue.However, the intrinsically fast cross-linking of PEG-4-arm-NHS mightlead to irregular sealing of the wound site (by inhomogeneous cross-linkingwith tissue) or poor fixation to tissue (by limited cross-linkingwith the collagen carrier) rendering this hemostat less effectivefor some surgical bleedings. Moreover, the hydrophilic nature of PEGcan also cause the polymer to be flushed away from the carrier duringhemorrhage, which leads to a poor hemostatic action. These potentialdrawbacks might be solved by modifying and fine-tuning the polymerarchitecture and properties. However, PEG has limited options fortailoring the degree of functionalization (only via the end groups)and polarity, which prompts further research on alternative polymerswith hemostatic activity.
Poly(2-oxazoline)s (or POx23−26) are promising polymers for biomedical applicationsdue to their versatile synthesis,27−29 favorable cytocompatibility,30−32 and promising excretability.33−37 In terms of polymer architecture and function, POx possesses importantadvantages over PEG-based systems when applied in hemostatic materials.First, cationic ring opening polymerization (CROP) allows for theintroduction of both functional side chains and end groups, whichis not easily achieved by anionic polymerization of PEG-based systems.Moreover, this polymerization technique allows for the synthesis ofa range of copolymers, which makes it possible to accurately controlthe polarity and degree of side-chain functionalization of the resultingpolymer.
In order to achieve optimal hemostatic performance,three mainaspects of the hemostatic device should be optimized, namely, (1)the carrier, (2) the polymer coating, and (3) the coating applicationmethod onto the carrier material. As a carrier, we selected a porousgelatin sponge. Although this carrier is animal derived, it has advantagesover other carrier materials, since it is fully biodegradable (4–8weeks), shows effective uptake of blood and is already registeredas a hemostatic product.13 Moreover, primaryamines are available in gelatin to allow for the formation of covalentcross-links between the carrier, blood proteins, and tissue in orderto create a gel that seals off the wound surface and stops the bleeding(Figure Figure11).
Schematic overviewof application method and mechanism of actionof poly(2-oxazoline) coated hemostatic patches. (A) Preparation ofhemostatic patch by spray-coating POx-NHS onto a gelatin sponge. (B)Application of the patch onto the wound site. (C) Hemostasis is obtainedby covalent cross-linking between the gelatin sponge, POx-NHS, bloodproteins, and tissue in order to create a gel which seals off thewound surface and stops the bleeding.
Regarding polymer design, for optimal hemostatic performance,thepolymer should have sufficient reactive moieties (NHS-esters) forcovalent cross-linking (e.g., with blood proteins). The polymer compositionshould furthermore be chosen in such a way that the polymers are solublein water (beneficial for their biological activity) and in organicsolvents (beneficial for polymer processing). Moreover, the reactivemoieties should be available for cross-linking, which requires reactiveside chains of sufficient flexibility and length as well as an overallpolymer composition which is polar enough to allow effective wettingunder physiological conditions. The cross-linking capacity shouldbe optimized to ensure that the polymer has sufficient time to cross-linkwith the various components (blood, carrier, and tissue).
Regardingthe coating of the hemostatic patch, we hypothesizedthat various parameters are important to achieve the desired hemostaticproperties. First, the reactive polymer should be equally distributedover the carrier in order to obtain homogeneous hemostatic propertiesover the whole area of the coated patch. Second, the polymer and carriershould be combined in such a way that undesired cross-linking duringthe coating process is prevented. Moreover, after coating, porosityshould be partially conserved in order to obtain a hemostatic patchwith a dual mechanism of action of both gelatin (natural coagulationcascade) and the reactive polymer (sealing off the wound site by covalentcross-linking). Moreover, the blood uptake of the coated patches shouldbe satisfactory to allow for cross-linking with all patch components(gelatin, reaction polymer, blood, and tissue), but also resistantenough to prevent excessive blood flow through the patch.
Inthis work, we demonstrate a versatile strategy for the preparationof a poly(2-oxazoline) based hemostatic device. First, a series ofNHS-ester functionalized POx (POx-NHS) with different ratios of NHSesters and polar groups was synthesized. We studied the capacity forcovalent cross-linking between these polymers and whole blood (hemostaticperformance) in order to correlate the hemostatic performance withthe polarity of the polymers (measured by contact angle measurements).With the preselected polymers, we utilized a spraying procedure tocreate a series of homogeneously coated patches. The coated patcheswere tested in vitro, for, for example, blood uptake and cross-linkingability. The best-performing patches in these tests were selectedto demonstrate in vivo efficacy in a compromised liver and spleeninjury model of profuse bleedings in heparinized pigs.
2. Materials and Methods
2.1. Materials
Gelatin sponges (GelitaRapid, origin: porcine, 5 × 8 × 0.2 cm) were obtained fromGelita Medical. Hemopatch was obtained from Baxter (Deerfield, IL,U.S.A.). Tachosil was obtained from Takeda (Linz, Austria). Pentaerythritoltetra(succinimidyloxysuccinyl) poly(ethylene oxide) (PEG-4-arm NHS)was obtained from NOF America corporation. Heparinized human wholeblood was obtained from Sanquin (Nijmegen, The Netherlands).
2.2. Synthesis
Experimental proceduresfor the synthesis of P1–P7 can befound in the Supporting Information.
2.3. Gelation Test
This experiment wasperformed using an inverted vial test adapted from literature.38 Polymer powders (20 mg) were mixed with freshlyobtained heparinized human whole blood (1 mL) in a glass vial andvortexed until a visible gel was formed (gelation time).
2.4. Contact Angle Measurements
Microscopecoverslips (2 cm2) were soaked in absolute ethanol, sonicated(30 s) and dried under reduced pressure for 15 min. Polymer filmswere prepared by spin-coating the polymer solutions (15 mg/mL in DCM,1 mL) onto the microscope coverslips (12000 rpm, 30 s) using a Spin150 spin-coater. Subsequently, the coated slides were dried overnightunder reduced pressure. Static contact angles were measured on anOCA-20 goniometer. For each measurement, 1 μL of doubly distilledwater was placed onto the spin-coated films at room temperature. Thespreading of the droplet was imaged using a high speed video camerausing 1 frame/second for 30 s. The contact angle was determined basedupon the Laplace Young fitting using the imaging software providedby the supplier (SCA 20, version 2.1.5 build 16). To determine thecontact angle, the first representative frame in which a drop shapewas observed was selected for analysis. The measurements were conductedin triplo per sample (n = 3).
2.5. CoatingDeposition
Spray coatingwas performed using an Exactacoat spraying machine (Sono-Tek) equippedwith an Accumist ultrasonically agitated nozzle. Coating was performedat a dispensing rate of 1 mL/min, a pressure of 40 mbar and a coatingspeed of 40 mm/s, by moving both in the xy-directionover a programmed area. The nozzle height was set 30 mm from the topof the substrate. The coating density was adjusted by spraying multiplelayers of polymer (coating cycles (n)) onto the substrate.Polymer solutions were prepared in 2-butanone/2-propanol (v/v, 1:1)with a final polymer concentration of 90 mg/mL. After coating, thepatches were dried in a vacuum oven (50 mbar, 50 °C). The coatingdensity (mg/cm2) was determined by weighing the patches(before (carrier) and after coating (carrier + polymer) (mg)) dividedby the coated area (cm2).
2.6. ScanningElectron Microscopy
Sampleswere attached to an aluminum holder by conducting carbon tape. Afterward,these samples were sputter coated using a gold/palladium coater (Cressington208 HR) for 30 s (80 mA). At different magnifications, images wereacquired at an accelerated voltage of 3 kV using a JEOL 6330 CryoField Emission Scanning Electron Microscope (SEM).
2.7. Blood Uptake
This experiment wasperformed using a procedure adapted from literature.13 Coated gelatin patches with different coating densities(0, 3, 6, and 9 mg/cm2) were weighed (“dry weight”(mg)) and soaked into a mixture of heparinized blood/PBS (v/v, 1:1)with the coated side facing the blood mixture. The patches were allowedto absorb blood for 30 s. After this, superficial blood was removedusing a filter paper and the patches were weighed again (“weightafter” (mg)) and the amount of absorbed blood (“blood”(mg)) was determined (wt after (mg) – wt before (mg)). Theblood uptake was defined by calculating the amount of absorbed bloodper g patch. The measurements were performed in 6-fold for each sample(n = 6). Significant differences between sampleswere analyzed using ANOVA followed by a post hoc Tukey-Kramer multicomparison test.
2.8. Adhesion Test
This experiment wasperformed and designed according to modified ASTM F2258–05standards.39 The samples were attachedwith double sided tape to 3D-printed grip tabs of 2 cm2 and these tabs were placed into a single column tensile tester (Z2,5,Zwick/Roell,Ulm, Germany, containing a 20 N load cell). Heparinized blood (200μL) was put between the coated patches which were pressed downusing a weight of 20 g. The patches were allowed to cross-link withblood for defined times (1, 5, and 15 min). Subsequently, the patcheswere pulled apart and the load at failure (Fmax, N) was measured. The measurements wereperformed in 6-fold for each sample (n = 6). Significantdifferences between samples were analyzed using ANOVA followed bya post-hoc Tukey-Kramer multicomparison test.
2.9. In VivoEfficacy Test
Heparinized(10k units) pigs (n = 4, 30 kg) were used in thisstudy. Permission for this experiment was granted by the responsibleethical committees at the Ministry of Education of the Czech Republic(Project #56–2015/processing #MSMT-42725/2015–6) Surgerywas performed using standard aseptic techniques. A midline laparotomywas performed to access liver and spleen. Using a biopsy punch, standardizedlesions were created in liver and spleen (8 mm diameter, 3 mm deep).Hemostatic patches of 2.7 × 2.7 cm were used in this study (n = 8 per prototype randomized per organ using a balancedlatin square). After the lesion was created, the blood flow was assessedaccording to a visual scoring system (0 = no bleeding, 0.5 = oozing,1 = very slight, 2 = slight, 3 = moderate, 4 = severe) described inliterature.40 Afterward, superficial bloodwas removed using a dry gauze. Subsequently, the patches were appliedwith the coated side facing the organ and digital pressure was appliedfor 1 min using a dry gauze. The efficacy of the patches was evaluatedafter 0, 1, and 5 min by monitoring the bleeding (yes or no). Successfulhemostasis was achieved if no bleeding was observed after 5 min withoutpressure (yes or no). Additionally, after 5 min, the bleeding wasscored according to the scoring system and the adhesion of the patchto the organ was tested.
2.10. Statistics
Statisticalanalyseswere conducted using GraphPad Instat software. All results were reportedas mean ± standard deviation. Differences among groups were analyzedby ANOVA using a Tukey-Kramer Multi comparison test and p-values of 0.05 or lower were considered as significantly different.
3. Results and Discussion
3.1. Synthesis
In order to create poly(2-oxazolines)with the desired characteristics for application as reactive coatingin a hemostatic patch, both polarity and reactivity had to be optimized.We selected NHS-esters as the reactive moieties in view of their reactivitytoward primary amines and their routine application in related medicaldevices.7 Since direct incorporation ofNHS-esters as functional group is not compatible with cationic ringopening polymerization (CROP), we used methyl ester functionalized2-methoxycarbonylethyl-2-oxazoline (MestOx) as monomer instead. Thisgroup can be easily modified after the polymerization by direct amidation41 or hydrolysis,42−44 as has been describedin literature. Furthermore, it has been efficiently copolymerizedbefore with various comonomers (including 2-ethyl-2-oxazoline (EtOx)45 and 2-n-propyl-2-oxazoline(nPropOx)46). For thesynthesis of the various POx-NHS (P1–P7) we used two different synthetic routes, as depicted in Scheme 1. In all cases, polymerswere synthesized by CROP of different ratios of EtOx, nPropOx, and MestOx under inert atmosphere using microwave conditions,47 yielding both nPropOx-MestOxand nPropOx-EtOx-MestOx copolymers. In the firstroute, the MestOx groups were hydrolyzed (0.1 M NaOH), resulting ina copolymer containing carboxylic acid moieties, which were subsequentlyactivated with N-hydroxysuccinimide yielding P1–P6. In the second route, MestOx waspostmodified by an amidation reaction with ethanol amine, yieldingcopolymers equipped with a hydroxyl moiety in the side chain. Subsequently,these hydroxyl groups were partially converted to carboxylic acidmoieties using succinic anhydride, which were subsequently modifiedinto reactive esters by coupling with N-hydroxysuccinimide(P7). Importantly, this second route installs a hydrolyticallysensitive group in the side chain, favorable for degradation. As listedin Table 1, polymers P1–P7 were synthesized with good controlover the ratio of functional groups, number-average molar mass anddispersity values. The various synthesized polymers (P1–P7) were analyzed with regard to the amountof NHS groups present using both 1H NMR and UV–visspectroscopy, confirming a good agreement between the theoreticaland experimental compositions.
Synthesis of NHS-Ester FunctionalizedPolymers (POx-NHS; P1–P7)Reagents and conditions: (i)methyl tosylate, 140 °C, CH3CN, (ii) 0.1 M NaOH, rt,(iii) NHS–OH, DIC, DCM, rt, (iv) 2-amino-ethanol, 60 °C,300 mbar, (v) succinic anhydride, DMAP, DMF/DCM (v/v, 1:9, rt).
Table 1
% funct. (1H NMR) | |||||||||
---|---|---|---|---|---|---|---|---|---|
m | n | x | UV | Mn (kg/mol) | |||||
# | polymer | m/n/x | nPropOx | EtOx | OH | NHS | NHS | SECa | Đa |
P1 | P(nPropOx-c-NHS) | 90–0–10 | 90 | 10 | 11 | 12.6 | 1.15 | ||
P2 | P(nPropOx-c-NHS) | 75–0–25 | 71 | 29 | 26 | 13.9 | 1.11 | ||
P3 | P(nPropOx-c-EtOx-NHS) | 40–50–10 | 40 | 49 | 11 | 9 | 12.4 | 1.18 | |
P4 | P(nPropOx-c-EtOx-NHS) | 40–35–25 | 40 | 36 | 24 | 22 | 12.4 | 1.26 | |
P5 | P(nPropOx-c-EtOx-NHS) | 50–40–10 | 49 | 40 | 11 | 11 | 12.3 | 1.16 | |
P6 | P(nPropOx-c-EtOx-NHS) | 50–25–25 | 50 | 26 | 24 | 23 | 14.6 | 1.18 | |
P7 | P(nPropOx-c-OH-NHS) | 70–10–20 | 70 | 15 | 15 | 15 | 18.8 | 1.25 |
aSEC was calibrated against PMMAstandards; eluent: 0.1% LiCl in DMA.
3.2. Hemostatic Performance
As a firstscreening for hemostatic activity, the POx-NHS polymers were broughtin contact with human whole blood and the formation of gels by mixingpolymers with blood was analyzed using the inverted vial test. Besidesthe POx-NHS series, negative controls (polymers without NHS ester)were tested as well. In addition, a benchmark polymer (PEG-4-arm NHSused in Hemopatch) was included as a positive control to compare functionalgroup density (mmol NHS/g polymer) in relation to the usage of differentpolymers. The results of these tests are listed in Table 2. As expected, polymers withNHS-esters (P1–P7) gelled with blooddue to the presence of the amine-reactive NHS-esters groups, unlikethe negative controls (P8–P12), whichdid not. Gelation times of the POx-NHS series (P1–P7) varied between 1 min (P7) to 6 min (P1–P2), which was slower than the PEG-4-armNHS benchmark polymer, which formed a gel with blood instantaneously.
Table 2
# | polymer | %NHS | functional group contenta (mmol NHS/g polymer) | contact angleb (deg) | gelation timec (min) |
---|---|---|---|---|---|
P1 | P(nPropOx-c-NHS) | 10 | 0.76 | 36 | 6 |
P2 | P(nPropOx-c-NHS) | 29 | 2.19 | 57 | 6 |
P3 | P(nPropOx-c-EtOx-NHS) | 11 | 0.89 | 26 | 3 |
P4 | P(nPropOx-c-EtOx-NHS) | 24 | 1.59 | 24 | 3 |
P5 | P(nPropOx-c-EtOx-NHS) | 11 | 0.88 | 23 | 3 |
P6 | P(nPropOx-c-EtOx-NHS) | 24 | 1.57 | 21 | 3 |
P7 | P(nPropOx-c-OH-NHS) | 15 | 0.91 | 23 | 1 |
Controls | |||||
P8 | P(EtOx) | 26 | no gel | ||
P9 | P(nPropOx) | 57 | no gel | ||
P10 | mPEG-OH | 24 | no gel | ||
P11 | PEG-4-arm-NHSd | 0.36 | 21 | instantaneous |
aCalculatedusing NHS-content, whichwas determined by 1H NMR spectroscopy.
bThe measurements were conductedin triplo, blank measurement glass slide (66°).
cThe gelation was determined by theinverted vial method.
As polarity was anticipated to be an important feature of the hemostaticcapacity of the polymers, POx films were spin-coated on glass slidesafter which static contact angle measurements were performed.48 Based on these contact angle measurements, itcan be concluded that polymers functionalized with hydrophilic groups(PEG, OH or EtOX) (P3–P8 + P10 and P11) exhibit contact angles in a similarhydrophilic range (21–26°), while polymers without hydrophilicgroups (P1 and P2 + P9) havehigher contact angles, thereby making a clear difference between hydrophilicand somewhat more hydrophobic copolymers. It was further calculatedthat the PEG-based control (P11) shows a much lower contentof NHS-functional groups (0.36 mmol/g polymer) compared to P1–P7, with values ranging from 0.76 mmol/g polymerfor P1 to 2.19 mmol/g polymer for P2, whichis a direct result of the limited functionalization possibilitiesof PEG via the end groups.
From both tests, it can be concludedthat NHS-esters are essentialfor the formation of chemical cross-links with blood proteins. However,having a surplus of NHS-esters does not result in faster gelation.POx-prototypes which contain both NHS-esters and hydrophilic groupsshow faster gelation (P3–P7) comparedto polymers without hydrophilic groups (P1 and P2), but slower than PEG-4arm-NHS (P11), whichcross-linked instantaneously. It was observed that the fast-gelatingpolymers also exhibited low contact angles. The difference in gelationspeed between PEG-4-arm NHS and POx-NHS prototypes could, however,not be explained from the contact angle measurements. We assume thatpolarity and mobility of the polymer chains (limited by the spacerlength between the polymer backbone and NHS-ester groups) are importantparameters. PEG-4-arm NHS shows the fastest gelation, since the NHS-estergroups are highly mobile because of their attachment to the hydrophilicchain ends of the PEG-polymer. Within the POx-NHS samples, P7 shows the fastest gelation (1 min) because it has a longer spacerbetween the NHS group and the polymer backbone compared to P1–P6. Finally, the differences between P1–P6, with the same spacer length, can be explainedbecause of polarity of the polymers; polymers containing hydrophilicEtOx groups (P3–P6) show gelationwithin 3 min, while polymers without these groups (P1 and P2) show gelation around 6 min. Due to its fastgelation, we selected P(nPropOx-OH-NHS) (P7) as the main candidate for further development of hemostatic patches.
3.3. Spray Coating Deposition
To coverthe gelatin carrier with a polymer (P7) coating, a procedurewas required that would result in a homogeneous polymer layer withoutcompromising the beneficial properties of the gelatin carrier in termsof, for example, blood uptake capacity. Therefore, we used an ultrasonicspraying technique to deposit the polymer from volatile organic solventsof low toxicity onto the gelatin sponge and tune the amount of polymerby coating multiple layers (coating cycles) followed by drying thecoated patches in a vacuum oven.
Using this approach, hemostaticpatches (G1–G4) were prepared atvarious coating densities (0–9 mg/cm2) using a polymersolution of P7 (90 mg/mL in 2-propanol/2-butanone (v/v,1:1)). We observed a linear relationship between the coating density(mg/cm2) and the amount of coating cycles (Table 3). Additionally, the coatedpatches were analyzed by scanning electron microscopy (SEM; Figure Figure22), which revealedthat the pores of the carrier were not sealed by the polymer coatingafter applying up to six coating cycles. Furthermore, the coatingwas homogeneously spread onto the carrier material, unlike Hemopatch(based on 4-arm-PEG), which showed a heterogeneous coverage revealingPEG-coated and uncoated domains. The analytical data of G1–G4 are summarized in Table 3. Importantly, as POx is functionalized witha higher number of NHS-esters than PEG, a lower amount of polymerwas required (5.7 mg/cm2 for G3) in orderto obtain a similar functional group density as Hemopatch (∼5.2μmol NHS/cm2), which is beneficial if an open, porousstructure is required for the carrier material.
SEM images of POx-NHScoated patches (G1–G4) and Hemopatch(PEG). Scale bars correspond to 1 mm or100 μm (bottom right picture).
Table 3
Coating and Functional Group Densitiesof Patches Prepared with P7 (G1–G4)
coating density (mg/cm2) | |||||
---|---|---|---|---|---|
samples | theoretical | measured | |||
# | mean | st dev | n | functionalgroup densitya (μmol NHS/cm2) | |
G1 | 0 | ||||
G2 | 3 | 3.06a | 0.01 | 3 | 2.80 |
G3 | 6 | 5.71a | 0.13 | 9 | 5.18 |
G4 | 9 | 9.22a | 0.01 | 3 | 8.36 |
Hemopatch (PEG) | 16.8b | 2.2 | 5 | 5.38 |
aMass differencebefore (gelatin)and after coating (gelatin + POx-NHS).
3.4. In Vitro Tests
3.4.1. BloodUptake
The blood uptake ofthe different POx-NHS coated patches (G1–G4) and Hemopatch was evaluated by soaking the patches (withthe coated side in contact with blood) in a mixture of blood/PBS for30 s and determining the blood uptake by weighing the carriers beforeand after the soaking process (Figure Figure33A). It was observed that the uptake capacity of thepatches was reduced with increasing coating density. Although thepolymer coating did not seal off the pores of the underlying gelatincarrier (Figure Figure22),the blood uptake was clearly compromised by the deposition of POx-NHSonto the patches. Hemopatch was included as well in these measurements,and showed significantly lower blood uptake values compared to G1–G3. Since blood uptake is necessaryfor satisfactory blood distribution throughout the patch and subsequentcross-linking, we concluded that G3 was the best-performingprototype in this test, as it allowed for more effective blood uptakecompared to G4 and Hemopatch, but still prevented bleedingthrough the patches, which was observed for G1 and G2.
(A, B) In vitro tests. (A) Blood uptake capacity as a functionof coating density (**P < 0.001, *P < 0.01); (B) In vitro adhesion test: (i) blood was applied betweenthe patches, (ii) the patches were allowed to cross-link for definedtime points (t1, t5, t15 min), (iii) the samples were placed in aZwick Roell tensile bench and a vertical force was applied until failure,(iv) results of the adhesion test (*P < 0.05,**P < 0.01, ***P < 0.001).
3.4.2. AdhesionTest
An in vitro adhesiontest was performed (according to ASTM F2258–05 standards) tostudy the attachment between the coated patches upon contact withblood (Figure Figure33B).The different patches (G1–G4) wereallowed to covalently cross-link onto each other for 1, 5, or 15 min,after which the adhesion force (N) was measured untilthe patches were separated. Both a negative control (G1, carrier without polymer) and a benchmark (Hemopatch) were includedin this study. The data demonstrated that the NHS-ester free blanksamples (G1) did not adhere to each other, as reflectedby adhesion forces of less than 0.5 N, which confirms that NHS-estergroups are necessary for the formation of covalent cross-links. Thecoated samples (G2–G4) showed anentirely different behavior. At 1 and 5 min contacting time, low adhesionforces were measured that were comparable to G1, indicatinga low degree of cross-linking. At 15 min, however, a 3-fold largerforce (1.5 N) was needed to separate both patches. This indicatesthat more cross-links are formed during the extended cross-linkingtime (15 min), resulting in larger adhesion forces. However, sincethese forces were in the same range for all three patches, it canbe concluded that coating density did not affect the extent of adhesionin this experiment. By testing Hemopatch, adhesion forces within 1and 5 min were similar to G2–G4 after15 min. We conclude that this product generally cross-links fast andforms strong gels with blood and carrier, which is in agreement withthe blood gelation tests. While the differences regarding adhesionforces between the POx-NHS samples (G2–G4) and Hemopatch (15 min) were statistically significant comparedto NHS-ester free G1 (15 min), adhesion forces after15 min were not statistically different between Hemopatch and thePOx-NHS samples (G2–G4). In summary,it can be concluded that Hemopatch cross-linked faster than POx-NHSsamples, whereas the final adhesion strength after 15 min was comparablefor both samples.
3.5. In Vivo Efficacy Test
The POx functionalizedpatches were also evaluated in a clinically relevant setting by usingan established in vivo pig model for profuse bleedings.40 In brief, standardized bleedings (8 mm diameter,3 mm deep) were created in the liver and spleen of heparinized pigs(n = 4, 30 kg, 10 k heparin). The bleedings wereimaged at selected time points (0, 1, and 5 min after creation ofthe bleeding) (Figure Figure44A) and the hemostatic efficacy of the different patches was assessedat 0, 1, and 5 min (bleeding/no bleeding). In addition, the bleedingscore after 5 min was assessed using a visual scoring system rangingfrom 0 (effective hemostasis) to 4 (severe bleeding) (Figure Figure44B). The efficacy of hemostasisof POx-NHS coatings was tested for G3, which had a similarfunctional group density asthe benchmark Hemopatch (∼5.2 mmol NHS/cm2; Table 3), but with a differentpolymer coating coverage. G1 was used as negative control(no coating). In addition, Tachosil (a collagen carrier coated withhuman derived fibrinogen and thrombin) was selected because of itscommon use during liver resections.15 Theresults of this study are depicted in Figure Figure44A,B. G3 was the best-performinghemostatic patch in this pig model; in 7 out of 8 events, hemostasiswas obtained (bleeding score after 5 min: 0, no bleeding; Figure Figure44B). In the remainingevent (1 out of 8), insufficient pressure during application resultedin poor hemostatic action (bleeding score: 2, slight bleeding) (Figure Figure44B). In all cases,no significant blood flow through the patch was observed using G3, as was expected from the blood uptake experiments. Evidently, G1 was not effective at all in this bleeding model and significantblood flow through the patch was observed in line with the in vitroblood uptake experiments (Figure Figure33A). Moreover, in none of the events hemostasis wasobtained, which can be related to the absence of chemical cross-linkers.As a result, using G1, in all events, severe bleedingswere scored after 5 min (bleeding score: 4; Figure Figure44B). In the experiments using Tachosil, onlyin 2 out of 8 events hemostasis was observed, whereas moderate bleedingswere scored for all other cases (Figure Figure44B). This poor hemostatic efficacy might bedue to the use of a high heparin dose (10 k units) in this pig model,which inhibits hemostasis solely based on the natural coagulationcascade. Using Hemopatch, effective hemostasis was obtained in 5 outof 8 events (Figure Figure44A), where bleeding scores varied from 0 (no bleeding) to 3 (moderatebleedings; Figure Figure44B). Generally, Hemopatch adhered well and quickly to the tissue,which made repositioning challenging, a trend that was observed inthe in vitro gelation tests as well. Unlike G3, slightbleeding at the edges of the patch was observed in cases where hemostasiswas not obtained (Figure Figure44), which is possibly related to the inhomogeneous depositionof the polymer coating compared to the POx-NHS coated patches (Figure Figure22). From this in vivostudy, it can be concluded that sealants that rely on chemical cross-linkingwith surrounding soft tissues and blood proteins (G3 andHemopatch) hold great promise for the treatment of profuse bleedingmodels, unlike patches which are solely dependent on the natural coagulationcascade (noncoated patch (G1) and Tachosil), which werenot effective in obtaining hemostasis in these models. Comparing G3 and Hemopatch, POx-NHS samples have the additional benefitthat they are coated more homogeneously than Hemopatch, which resultsin a uniform sealing of the wound site. In addition, POx-NHS samplesare easier to handle due to their slower adhesion, which allows repositioningof the patch if required.
In vivo study on pig spleen. (A) Images of thedifferent prototypesat selected time points (0, 1, and 5 min), including the success rateof hemostasis. (B) Bleeding scores after 5 min according to the scoringsystem40
4. Conclusions
In this work, we have successfullydeveloped a hemostatic devicebased on NHS-ester functionalized POx coated on a gelatin patch. Weobserved that the polymer should contain both NHS-esters as well ashydrophilic groups to ensure optimal hemostatic performance. Furthermore,we found that coating homogeneity and density are crucial parametersin order achieve the desired hemostatic action in vitro (measuredby adhesion tests) as well as the desired amount of blood uptake.In vivo efficacy tests in a compromised pig model using heparin demonstratedthat POx-NHS coated patches displayed a similar hemostatic efficacyas compared to Hemopatch. POx-NHS patches were superior to productsrelying on activation of the natural coagulation cascade. In contrastto PEG, the structural versatility of POx allows further fine-tuningof the hemostatic performance, thereby rendering POx-NHS polymersexcellent candidates for further development of hemostatic patches.
Acknowledgments
This work was supported by The Netherlands Institute for RegenerativeMedicine (NIRM, Grant No. FES0908), NWO (KIEM 731.013.107, LIFT 731.015.415),Europees Fonds voor Regionale Ontwikkeling (EFRO 2011-014237) andGATT Technologies bv. The authors would like to thank Elvy de Hoog(Radboud University) for help with the polymer synthesis, Paul Riedel(Rubroeder GmbH) for assisting with the spraying experiments, RogerLomme (Department of Surgery (Radboud University medical Center) forassisting with the adhesion experiments and in vivo study, and Elsvan der Leyden (Ghent University) for the assisting with the contactangle measurements.
Supporting Information Available
The SupportingInformationis available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00683.
- Description ofthe materials which are used and theexperimental procedures of the monomers, intermediate products, andfinal polymers (P1–P7) are describedin S1. 1H NMR spectra of both P3–P6 and P7, can be found in S2 and S3, respectively(PDF).
Notes
The authors declare nocompeting financial interest.
Supplementary Material
References
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