Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX-XXX
pubs.acs.org/journal/abseba
Absorbable Hemostatic Aggregates Allen Y. Wang,*,† Joseph Rafalko,∥ Melinda MacDonald,‡ Xintian Ming,§ and Richard Kocharian# †
Biosurgery R&D, ∥Analytical Characterization, §Product Microbiology, and #Medical Affairs, ETHICON, Inc., Johnson & Johnson Medical Devices Companies, Route 22 West, Somerville, New Jersey 08876-0151, United States ‡ Preclinical Center of Excellence, Johnson & Johnson Medical Devices Companies, Route 22 West, Somerville, New Jersey 08876-0151, United States S Supporting Information *
ABSTRACT: Topical absorbable hemostats are routinely utilized in surgical procedures to assist in controlling intraoperative bleeding. SURGICEL Original Absorbable Hemostat, one of the most frequently used adjunctive hemostats, is composed of oxidized regenerated cellulose (ORC). We report here that a novel powdered form of ORC, composed of aggregates of ORC fine fibers, provides additional valuable hemostatic performance characteristics and retains the biochemical and bactericidal profile of the parent ORC fabric. The ORC aggregates are more effective in promoting coagulation than their constituent ORC fine fibers because of more favorable surface energetics and surface area. Aggregates with similar particle size distributions that have higher sphericity values exhibit better coagulation efficacy. Finally, ORC aggregates more effectively promote clot formation than starch-based hemostatic particles. The results of this investigation indicate that the efficacy of this novel powdered hemostat is based on its chemical composition, morphology, and particle surface energetics. KEYWORDS: hemostat, oxidized regenerated cellulose, powder, surface energy, bactericidal increased rates of morbidity.4,5 These types of bleeding situations vary from discrete to diffuse and from a small to large surface area, as well as the source of bleeding that can be arterial/high pressure or venous/low pressure. In addition, the bleeding site may be easily accessible, or it may originate from an anatomical location that is difficult to visualize and access. Therefore, the selection of an optimal method of hemostasis or topical hemostatic agent is critical in order to achieve complete and sustained hemostasis. Conventional methods used to achieve hemostasis include the use of a variety of surgical techniques including sutures, ligature clips, and energy-based devices.3 When these surgical measures are ineffective or impractical, adjunctive hemostatic techniques and materials are routinely used to control the bleeding. Adjunctive hemostats can be categorized into two major groups based on their mode of action: the ones that
1. INTRODUCTION Incomplete hemostasis during surgery can adversely affect patient outcomes, hospital costs, and resources. This is increasingly important because of the growing number of compromised patients that are presenting for surgery every day. These patients present with numerous comorbidities such as uncontrolled diabetes, obesity, and certain types of cancer requiring extensive chemotherapy. At the same time, the number of older patients who are on chronic anticoagulant/ antiplatelet therapies that are undergoing surgery is increasing, all of which can increase the risk of intraoperative bleeding and the incidence of associated complications. Bleeding occurs routinely during surgical procedures and still remains a major cause of death in trauma patients.1 When intraoperative bleeding is relatively minor, it can often be controlled by a normally functioning coagulation system with application of direct pressure. In cases of more intensive bleeding, adjunctive hemostatic agents may be required to minimize blood loss.2,3 Excessive blood loss necessitates blood or blood product transfusions, which in turn are associated with © XXXX American Chemical Society
Received: June 22, 2017 Accepted: October 24, 2017
A
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
2.2. Scanning Electron Microscopy. The specimens of ORC aggregates and blood were fixed with 4% glutaraldehyde in phosphatebuffered saline (pH 7.3) (PBS) and stored at 2−8 °C overnight. The test articles were removed from the vial and rinsed with PBS several times. They were then dehydrated by serial washes for 30 min × 3 in increasing concentrations of ethanol (10%, 20%, 40%, 60%, 80%, 100%), and then dried with hexamethyldisilazane in a fume hood. Each test article was mounted on a carbon stub using carbon paint, followed by gold sputtering to render the test article conductive. The prepared test articles were examined by scanning electron microscopy using a JEOL JSM-5900 LV at 5KV under high vacuum at a 2.0−2.5 cm working distance. 2.3. Optical Microscopy. Test articles were imaged before and after addition of saline solution (0.9% Sodium Chloride Irrigation, USP, Baxter Healthcare Corporations) with a Keyence VHX-600 digital microscope. The dry test articles were placed on glass slides covered by coverslips and photographed. Then 100 μL of saline was added to the dry test articles, which were incubated at room temperature for 2 min, and photographed again. The optical microscopic images of ORC aggregates were taken by a Carl Zeiss SteREO Discovery systemV20 and were analyzed with AxioVision SE64 imaging software. 2.4. Infrared Spectroscopy. Fourier transform infrared spectroscopy was performed on a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). 2.5. Carboxyl Content. The carboxyl content was determined according to methods specified in the 2016 USP Monograph 39, 3053−3054.16 2.6. Size and Shape Determination. Particle sizes and shapes were obtained with a Sympatec QICPIC image analyzer (Sympatec GMBH, Clausthal-Zellerfield, Germany). It has a camera resolution of 1024 × 1024 pixels with a pixel size of 10 × 10 μm. Its measurement range is from 5 to 1705 μm. A VIBRI/L vibratory feeder was used to introduce solid particles into a RODOS/L disperser. Images of the dispersed particles were then obtained in the QICPIC with a camera frame rate of 450 fps. A Feret min Q3 method was used to calculate the particle sizes of the aggregates while a Sympatec LEFI Q1 algorithm was used to determine the fiber lengths of the fibers. 2.7. Sphericity. The sphericity [Sh(50)] of the median-diameter aggregates was determined by the Sympatec QICPIC method using the ratio of the perimeter P of the equivalent circle (PEQPC) to the real perimeter (Preal), in which A = area of the particle, shown in the equation Sphericity = (PEQPC)/(Preal) = 2(πA)1/2/(Preal). The area of the equivalent projection circle has the same area as the projection area of the real particle. We were able to control the sphericity and particle size distribution by adjusting the proprietary processing methods, shown in Table S1. 2.8. Surface Area and Surface Energy. Surface area and surface energy analyses were performed with inverse gas chromatography (Surface Measurement Systems Model IGC-SEA, Alperton, UK). Approximately 750 mg of each sample was packed into individual silanized glass columns (300 mm long by 4 mm inner diameter). Each column was conditioned with helium gas for 60 min at 37 °C and 0% relative humidity. All experiments were conducted at 37 °C, with 10 mL/min total flow rate of helium, using methane for dead volume corrections. The Brunauer, Emmett, and Teller (BET) model was used for surface area determinations,17 based on sorption isotherms with HPLC-grade decane (Sigma-Aldrich, St Louis, MO, USA) using the chromatograph in pulse sorption method.18−22 The surface energy profile was determined by mapping techniques in which the specific free energies of desorption were determined by polarization.23−25 The dispersive surface energy component (γD) was measured by the method of Dorris and Gray using nonpolar HPLC grade probes: decane, nonane, octane, and heptane (Sigma-Aldrich, St Louis, MO, USA).26 The specific free energy of adsorption (ΔGSP) was determined with HPLC-grade polar probes: acetone, ethanol, acetonitrile, ethyl acetate, and dichloromethane (Sigma-Aldrich, St Louis, MO, USA). The acid−base surface energy component (γsAB) was determined using the Good-van Oss-Chaudhury (GvOC)
provide a matrix to accelerate the patient’s natural coagulation cascade, and those that contain active biologic components such as thrombin and/or fibrinogen that will allow them to achieve hemostasis regardless of the patients’ coagulation status. The group of hemostats that assist in the coagulation cascade is also referred to as the adjunctive topical absorbable hemostats, which includes products based on oxidized cellulose (OC), oxidized regenerated cellulose (ORC), gelatin, collagen, chitin, chitosan, and polysaccharides.3 One of the longstanding and most frequently used topical hemostatic agents is a loose-knit fabric made from ORC known as SURGICEL Original Absorbable Hemostat. With a history of more than 50 years of clinical use, this brand of products has predominantly been used to assist in achieving and accelerating hemostasis when oozing or mild to moderate bleeding was observed in surgery. These types of products are optimal when bleeding is confined, because the product can be placed on the source of bleeding with manual compression to facilitate hemostasis. However, continuous oozing from large friable and raw surfaces can cause delays and surgeon frustration during open and endoscopic procedures. These situations require a hemostatic product that is ready to use with minimal to no preparation time. In addition, the product must be able to achieve quick coverage on the bleeding surface and the ability to achieve hemostasis with and without applying pressure. Currently, there are few powdered versions of absorbable hemostats in the market; however, although they are easy to use with minimal preparation time, their inability to act effectively in the wet field because of floating on the bleeding surface makes them relatively ineffective in achieving complete and durable hemostasis on broad bleeding areas. To address this need, we developed a powdered form of ORC that can work effectively on a broad surface in a wet field with the ability to form an adherent and durable blood clot. In this report, we describe a powder of optimized ORC hemostatic particles that is developed by producing fibrillar structured aggregates of ORC fine fibers. Various aggregation processes are used routinely in the food and pharmaceutical industries to improve the physical attributes of powders, including powder structure, particle size distribution, wettability, flowability, dispersibility, and sinkability.6,7 However, aggregation processes have not been used previously for production of hemostatic powders or related medical device products. There are many development challenges, including that although ORC is a highly effective hemostatic agent, its fibers have low crystallinity8 and are difficult to process mechanically. We are able to overcome these issues by processing ORC fibers through a series of innovative methods, and this report will describe how the blood clotting efficacy of ORC fibers can be improved by forming ORC aggregates.
2. EXPERIMENTAL SECTION 2.1. Test Materials. The topical absorbable hemostats used in this study were Arista AH (abbreviated as starch-based spheres; Bard Davol, Warwick, RI, USA); PerClot PHS (abbreviated as PC; CryoLife, Kennesaw, GA, USA); HemoCer (abbreviated as HC; BioCer Entwicklungs-GmbH, Germany); Traumastem Powder (abbreviated as OC for Oxidized Cellulose; Bioster AS, Veverska Bityska, Czech Republic); Equicel Powder (abbreviated as OCC for Oxidized Cotton Cellulose; Equimedical BV, Zwanenburg, Netherlands); ORC aggregates (Ethicon Inc., Somerville, NJ, USA); SURGICEL Original Absorbable Hemostat (abbreviated as ORC Original; Ethicon Inc., Somerville, NJ, USA). B
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Test materials were applied at 3, 5, 7 mg/200 μL except those in the Ccontrol group. The ratio of the test materials to blood was evaluated prior to executing this. The following ratios were evaluated: 1 mg/200 μL, 10 mg/200 μL, and 15 mg/200 μL. The 1 mg/200 μL did not meet the minimum sensitivity threshold for detection of coagulation while the 10 mg/200 μL and 15 mg/200 μL resulted in equipment error messages. As such, the 3−7 mg/200 μL ratios of test materials to blood were selected as the ratios required to adequately capture clotting performance when measured with the coagulometer. Citrated human blood was removed from the storage, allowed to warm up, and gently shaken using an orbital rotator at room temperature for 60 min prior to testing. The citrated human blood was purchased from Lampire Biological Laboratories, Pipersville, PA, USA. The blood was aseptically drawn from a normal, healthy, drug-free male donor who showed no signs of clotting deficiency. Calibration was performed on the benchtop coagulometer prior to testing, and followed a manual of Neoplastine CI PLUS STA. The PT time of abnormal control was obtained between 33 and 48 s, and the PT time of normal between 11.5 and 15.5 s. 0.02 M CaCl2 saline stock solution [1.33 μL of 1 M standard CaCl2 (Fluka Analytical) solution in 65.33uL saline (0.9% Sodium Chloride Irrigation, USP, Baxter Healthcare Corporations] was prepared. 66.67 μL of 0.02 M CaCl2 saline stock solution was added to each cuvette containing 1 iron ball and cuvettes were placed in the incubation area for prewarming at 37 °C for at least 60 s; 133.33 μL of the citrated human blood was then added to the cuvette, followed by immediately adding a preweighed test material via a micro funnel (QOSMEDIX 20038, 0.78 in. × 0.83 in.) into each cuvette and pressing start button to initiate test. No test material was added to blood for the control group. Clotting time was recorded for each application. 2.10. In Vivo Hemostasis. A pilot stability study was conducted evaluating the effects of product aging on the hemostatic performance of ORC aggregates. A swine acute liver punch biopsy model was used to evaluate hemostatic performance under controlled conditions. Within the larger product stability study, it was possible to detect effects of ORC aggregate sphericity on hemostatic efficacy because the older prototype samples had a lower sphericity [Sh(50) = 0.56], whereas more recently manufactured samples had a higher sphericity [Sh(50) = 0.76]. All test animals were handled and maintained in accordance with the current standards promulgated in the Guide for the Care and Use of Laboratory Animals.30 All aspects of the study were reviewed and approved by the ETHICON Institutional Animal Care and Use Committee. Animals were maintained under general anesthesia for the duration of the testing period and humanely euthanatized while under anesthesia at the completion of testing. Five female Yorkshire Cross pigs weighing 54−57 kg were included in the study. The pig is considered to be an appropriate surgical model mimicking human anatomy, physiology, blood volume, and tissuehandling properties that are essential to product application and assessment. Animals were positioned in a dorsal recumbency, and a ventral laparotomy was performed. The liver was located, incrementally externalized as needed, and kept moist with saline-soaked gauze. A 6 mm biopsy punch was used to incise the parenchymal surface of the liver at an angle perpendicular to the tissue to a depth stop of approximately 3 mm. The tissue in the center of the biopsy site was removed using forceps and surgical scissors. The defect was blotted with gauze and the assigned test article was applied to the site. A dry nonadherent wound dressing (Telfa nonadherent dressing, Kendall, Covidien, Tyco Healthcare, Massachusetts, USA) was applied over the test material, followed by digital pressure that was initially held for 30 s, followed by removal of the nonadherent dressing and a 30 s evaluation for hemostasis. When bleeding occurred during the initial evaluation period, pressure was immediately reapplied using a nonadherent wound dressing for an additional 30 s, followed by another 30 s evaluation for hemostasis up to a total allowable time of 2 min to achieve hemostasis. When bleeding did not occur within the 30 s observation period, the time to hemostasis was noted as the time when the last applied tamponade was released. Any site that achieved
model,27,28 in which the acid−base component is taken as the geometric mean of the Lewis acid parameter (γs−) and Lewis base parameter (γs+). The total surface energy (γT) is the sum of the dispersive surface energy and the acid−base surface energy (γT = γD + γsAB). The wettability or hydrophilicity of the test materials was determined by dividing the acid−base surface energy by the total surface energy (γAB/γT).11,12 The thermodynamic work of adhesion (Wad) for particle−blood interactions was compared to the work of cohesion (Wcoh) for particle−particle interactions for all hemostatic products based on the surface energy values of the individual components as shown below.
Wad = 2(γparticle Dγblood D)1/2 + 2(γparticle ABγblood AB)1/2
(1)
Wcoh = 2(γparticle Dγparticle D)1/2 + 2(γparticle ABγparticle AB)1/2
(2)
In the above equations, Wad is the work of adhesion, Wcoh is the work of cohesion, γD is the dispersive surface energy, and γAB is the acid− base surface energy. Because γbloodAB values were not available, the above equations were simplified to calculate the works of adhesion and cohesion from the total surface energy values only, using the surface tension value for blood (γbloodT) at 37 °C = 52.6 mJ/m2 based on the literature.29
Wad = 2(γparticle Tγblood T)1/2
(3)
Wcoh = 2(γparticle Tγparticle T)1/2
(4)
2.9. In Vitro Clotting Performance Evaluations. Two test methods were used to characterize clotting activity of the test materials with blood. In vitro clotting performance was determined by the percent of clotted porcine blood (by weight) retained in a vial following topical application of test materials. Each test powder article was gently mixed prior to testing. Blood was warmed at room temperature for approximately 60 min prior to testing. A 1 mL aliquot of blood was then transferred into a 2 mL glass vial, after which 100 mg of test article was applied to the surface of the blood through a microfunnel. Clotting was allowed to proceed for 2 min at room temperature. A cap was applied onto the vial. The capped vial was then flipped upside down and placed on the tap density analyzer (Quantachrome Autotap EC148; Quantachrome Instruments, Boynton Beach, FL, USA) and mechanically tapped 5 times (to remove access materials), and followed by vortexing for approximately 5 s (to avoid false clotting due to gel blocking effect). After 2 min, the cap was removed, and unclotted material was allowed to drain by gravity for 1 min. After dabbing with a piece of 3-fold paper towel (Uline S7127), the remaining residue in each sample was weighed. Weights of the samples were recorded throughout the mixing and dabbing process. Fresh porcine blood was collected in 4.5 mL glass Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), with a 3.2% buffered sodium citrate solution. Blood collection was reviewed and approved by the Ethicon Animal Use and Care Committee. Blood was collected from the cranial vena cava of adult swine that were under general anesthesia for unrelated study purposes. Blood was either drawn directly into a citrated glass venous blood collection tube (BD Vacutainer 4.5 mL glass plasma tube containing citrate solution, 0.5 mL; sodium citrate, 12.35 mg; citric acid, 2.21 mg (equivalent to 3.2% sodium citrate)), or alternatively blood was drawn into a sterile syringe and immediately transferred to the same type of tube. The blood sample was immediately inverted 4−5 times and placed under controlled refrigerated storage at 2−8 °C. The blood sample was moved to room temperature and inverted gently until the blood was completely homogeneous by visual observation before testing. In vitro clotting/gelation performance was determined by an electromechanical clot detection method (viscosity-based detection system) to measure the clotting/gelation times for various weights of the test materials with recalcified citrated human blood. This in vitro clotting/gelation performance was evaluated with the use of a coagulometer (Diagnostica Stago ST4 coagulation analyzer). C
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 1. Micrographs of ORC fine fibers and aggregates. Images A and B are scanning electron micrographs of ORC fine fibers and single ORC aggregate, respectively. Images C and D are light micrographs of an aggregate before and after immersion in saline solution. Images E and F are optical micrographs of ORC aggregates Sh(50) = 0.76 and ORC aggregates Sh(50) = 0.51, respectively. Branhamella; reinforced clostridial agar and broth (Hardy Diagnostics, Santa Maria, CA, USA) used for Clostridium and Bacteroides f ragilis; and Middlebrook 7H9 broth and agar (Difco, Franklin Lakes, NJ, USA) used with Mycobacterium phlei. Bactericidal efficacy was defined, per Clinical and Laboratory Standards Institute (formerly NCCLS) Guideline M-26A, as more than a 3-log reduction from the inoculum.31 The organisms tested against ORC aggregates were: Staphylococcus aureus (MRSA) ATCC 33591; Staphylococcus epidermidis (MRSE) ATCC 51625; Streptococcus pneumoniae (PRSP) OC 21411; Enterococcus faecium (VRE) ATCC 700221; Bacillus subtilis ATCC 6633; Bacteroides f ragilis ATCC 25285; Branhamella catarrhalis ATCC 25238; Clostridium perfringens ATCC 3624; Clostridium tetani ATCC 19406; Corynebacterium xerosis ATCC 373; Enterococcus cloacae ATCC 13047; Enterococcus faecalis ATCC 29212; Escherichia coli ATCC 8739; Klebsiella pneumoniae ATCC 10031; Micrococcus luteus ATCC 4698; Mycobacterium phlei ATCC 11758; Proteus mirabilis ATCC 12453; Proteus vulgaris ATCC 13315; Pseudomonas aeruginosa ATCC 9027; Pseudomonas stutzeri ATCC 17588; Salmonella enteritidis ATCC
hemostasis within 2 min was then lavaged with up to 10 mL of saline and observed for persistent hemostasis over another 30 s observation period. 2.11. In Vitro Bactericidal Efficacy. The in vitro bactericidal efficacy of ORC aggregates was determined in nutrient broth and then compared with ORC original and competing topical absorbable hemostats. Broth cultures were prepared by inoculation in 20 mL tubes of broth with a known number of viable organisms. The inoculated broth was divided into two equal parts; one part was treated with 2.5% w/v hemostat, the other was an untreated control. Viable counts were then performed by serial dilution and plating on solid media at time 0 and after the 24 h incubation. Aerobic organisms were incubated at 37 °C for 24 h. Anaerobic organisms were incubated for 5 days at 37 °C in an anaerobic chamber with an AnaeroPack system (ThermoFisher Scientific, Lenexa, KS, USA). The culture medium was typically tryptic soy broth or tryptic soy agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The exceptions were: brain-heart infusion agar and broth (BBL, Franklin Lakes, NJ, USA) used for Streptococcus, Corynebacterium, and D
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering 13076; Serratia marcescens ATCC 13880; Shigella sonnei ATCC 9290; Staphylococcus aureus ATCC 6538; Staphylococcus epidermidis ATCC 14990; Streptococcus agalactiae ATCC 624; Streptococcus pyogenes ATCC 12384; Streptococcus salivarius ATCC 13419; Lactobacillus rhamnosus ATCC 7469. In the comparative testing, the organisms used were: Staphylococcus aureus (MRSA) ATCC 33593; Escherichia coli ATCC 25922; Enterococcus faecium (VRE) ATCC 700221; Enterococcus faecalis ATCC 700802/v538. The test articles were: Starch-based spheres = Arista AH; PC = PerClot PHS; HC = HemoCer; OC = Traumastem; OCC = Equicel Powder; ORC aggregates; ORC Original = SURGICEL Original Absorbable Hemostat. 2.12. Statistical Analysis. Results are presented as means ± standard deviation. Data were analyzed using ANOVA in Minitab 17 Statistical Software. Statistical significance was considered achieved when P values were less than 0.05.
therefore the extent of hydrophilic or hydrophobic behavior of a material when mixed with whole blood. The Brunauer, Emmett, and Teller (BET) surface areas for high sphericity ORC aggregates [Sh(50) = 0.76], low sphericity ORC aggregates [Sh(50) = 0.51], ORC fine fibers, and starch based spheres are shown in Table 1. ORC aggregates with
3. RESULTS AND DISCUSSION 3.1. Physical and Chemical Composition of ORC Aggregates. A powder consisting of ORC aggregates was produced from the fabric form of SURGICEL Original Hemostat. The fabric form was taken through a series of proprietary processes during which the physical and chemical properties of the aggregates were carefully controlled. During the process of powder production ORC fine fibers (Figure 1A) are processed to form nearly spherical ORC aggregates (Figure 1B, C). Each ORC aggregate is composed of a number of ORC fine fibers that are interconnected at discrete points, forming an interlocking web without loss of the original fibrillar structure. The preservation of the fibrillar structure in the aggregates can be confirmed by deconstructing the aggregates in saline solution. Figure 1D shows an image of an aggregate after they have been in saline solution for a few minutes revealing that the individual constituent fibers are preserved. Figure 1E, F show the optical micrographs of ORC aggregates Sh(50) = 0.76 and ORC aggregates Sh(50) = 0.51, respectively. Particle size distributions were obtained for the ORC fine fibers and ORC aggregates using a Sympatec QICPIC image analyzer. Typical ORC fine fibers had length weighted fiber length D(10), D(50), and D(90) values of 30, 72, and 128 μm, respectively. A typical ORC aggregate had volume weighted Feret minimum diameter D(15), D(50), and D(90) values of 111, 178, and 307 μm, respectively, and a sphericity Sh(50) value of 0.76. The chemical structure of the ORC fine fibers and the ORC aggregates were analyzed to determine if there were any chemical changes induced during processing. The Fourier transform infrared (FTIR) spectra of the ORC fine fibers and ORC aggregates were virtually identical, indicating that no chemical changes occurred during processing. Furthermore, their carboxyl contents, as determined using USP methods, were very similar. The results of these tests indicated that ORC fine fibers and ORC aggregates are essentially chemically identical. 3.2. Surface Area and Surface Energy. To explore the observed in vitro clotting behaviors of the high sphericity ORC aggregates [Sh(50) = 0.76], low sphericity ORC aggregates [Sh(50) = 0.51], ORC fine fibers, and starch-based spheres in terms of their biochemical and biophysical properties, we measured the surface area, wettability, and surface energy of each hemostatic material. Both surface area and surface energy are known to affect cohesion and dispersion of particles. Wettability provides a relative measure of surface polarity, and
sphericity values of 0.51 and 0.76 had surface areas of 0.67 and 0.40 m2/g, respectively. ORC aggregates and fine fibers are composed of chemically identical oxidized regenerated cellulose, but ORC aggregates had a lower surface area/mass ratio because of their larger particle sizes. It was also determined that ORC aggregates with lower sphericity values had higher surface areas than aggregates with higher sphericity values despite having similar particle size distributions. In contrast, the starch-based spheres had the highest surface area of the four test materials. Surface energetics were measured for each of the materials at various surface coverages by inverse gas chromatography. The surface coverage is defined by n/nm where n is the concentration of the probe molecule and nm refers to the monolayer capacity of the probe molecule. The total surface energy, dispersive energy, acid−base energy, and wettability profiles for the materials are shown in Figure 2A−D. The total surface energy is the sum of the dispersive and acid−base surface energies. Wettability profiles were determined by dividing the acid−base surface energy by the total surface energy.11,12 Specific values for these energies measured at 0.01 n/nm surface coverage are listed in Table 2. The measurements at this low surface coverage are indicative of the energetics upon initial contact. Table 2 also lists the ratio of the work of adhesion (Wad) to the work of cohesion (Wcoh). This ratio gives an indication of the balance of forces between particle−blood (adhesion) and particle−particle (cohesion) interactions. For instance, if particle−particle interactions are too high, resulting in a high Wcoh value and low Wad/Wcoh ratio, then agglomeration, poor particle dispersion, or decreased particle−blood interactions may occur. Also, if the particle−blood interactions are too low, resulting in a low Wad and low Wad/Wcoh ratio, then poor particle−blood interfacial adhesion may occur. The equations for the work of adhesion and the work of cohesion are shown in the Experimental Section. 3.3. In Vitro Clotting Performance Evaluations. Two test methods were used to characterize clotting activity. ORC aggregates with similar particle size distributions prepared with 2 different sphericity values [Sh(50) = 0.51 and Sh(50) = 0.76] were evaluated. In addition, ORC fine fibers from which the aggregates are derived, and a powdered hemostat composed of starch-based spheres (Arista AH [Bard Davol, Warwick, RI, USA]) with a sphericity value of [Sh(50) = 0.93] were also evaluated. These in vitro investigations were initiated to determine how sphericity of the ORC test materials affected clotting and also how the performance of these ORC test
Table 1. Sphericity and Surface Area of Test Materials
E
test material
sphericity Sh(50)
surface area (m2/g)
ORC aggregates ORC aggregates ORC fine fibers Starch-based spheres
0.76 0.51 N.A. 0.93
0.40 0.67 1.17 2.03
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 2. Surface energy profiles for absorbable hemostats. The materials tested were: ORC aggregates at Sh(50) = 0.76 and Sh(50) = 0.51, ORC fine fibers, and starch-based spheres. All values were measured at 37 °C. Panel A is total surface energy; panel B is dispersive surface energy; panel C is the acid−base energy; panel D is the wettability profile.
Table 2. Surface Energetics Measured at 0.01 n/nm Surface Coverage test materials ORC aggregates Sh(50) = 0.76 ORC aggregates Sh(50) = 0.51 ORC fine fibers starch-based spheres
dispersive surface energy (mJ/m2)
acid−base surface energy ([mJ/m2)
total surface energy (mJ/m2)
surface wettability
ratio of work of adhesion to work of cohesion (Wad/Wcob)
48.4
1.9
50.4
0.0384
1.02
50.5
4.07
54.5
0.0746
0.982
44.6 83.8
0.110 0.130
1.08 0.792
39.7 72.8
4.9 10.9
materials compared with the performance of a starch-based spheres. The first in vitro clotting test measured percent clotting by weighing the amount of citrated porcine blood remaining in a tube after the addition of the hemostat and inversion of the tube. Test and commercial materials were graded on a 0 to 4 scale (minimum to maximum) for ability to penetrate the surface of the blood sample (100 mg/1 mL in a vial), and subsequent ability to induce blood clotting. Figure 3A−D shows blood samples prior to (panel A) through 2 min after addition of 100 mg of each test or commercial material (panel D). In each panel, tube #1 illustrates an untreated control, tube #2 was treated with starch-based spheres, tube #3 was treated with ORC fine fibers, tube #4 was treated with low sphericity ORC aggregates [Sh(50) = 0.51], and tube #5 was treated with high sphericity ORC aggregates [Sh(50) = 0.76]. Within seconds (Figure 3B, C) there were visible differences in the blood-penetrating activity of the different materials. The ORC aggregates with high sphericity [Sh(50) = 0.76] immediately penetrated the surface of the blood and initiated formation of a gelatinous mass that accelerated blood clotting (Grade 4). The ORC aggregates with less sphericity [Sh(50) =
0.51] penetrated, but to a lesser extent (Grade 3). In contrast, a large proportion of the ORC fine fibers (essentially aspherical) remained on the surface of the blood (Grade 2). The starchbased spheres remained on top of the blood demonstrating minimal surface penetration (Grade 1). This observation indicates that higher sphericity contributes to the bloodpenetrating characteristics of ORC aggregates. However, sphericity itself was not the only factor affecting penetration, as the starch-based spheres were the most spherical material tested [Sh(50) = 0.93] but showed the least penetration of the blood surface. Several other factors have been identified that also affect blood penetration and subsequent coagulation and these will be discussed later in this report. After 2 min, there were visible differences in the clotting activity of the different materials. The blood in tube #5 treated with high sphericity ORC aggregates was fully clotted (Grade 4), evidenced by the dark reddish-black color that is characteristic of blood coagulated with an ORC product. The blood treated with low sphericity ORC aggregates (tube #4) and ORC fine fibers (tube #3) was less effectively clotted (Grade 2), and the blood treated with starch-based spheres (tube #2) showed little evidence of clotting (Grade 1), F
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 3. In vitro clotting observations. From left to right, sample tubes contain untreated control blood (tube #1); starch-based spheres (tube #2); ORC fine fibers (tube #3); ORC aggregates Sh(50) = 0.51 (tube #4); and ORC aggregates Sh(50) = 0.76 (tube #5). Panel A shows blood before addition of the test materials. Panel B is a low-angle view and panel C is a high-angle view of the tubes within 10 s after addition of the test materials. Note that the high sphericity ORC aggregates in tube #5 have already penetrated the surface of the blood and have begun to initiate clotting. Low sphericity ORC aggregates in tube #4 remain somewhat superficial but have also initiated clotting. ORC fine fibers in tube #3 and starch-based spheres in tube #2 remained on the surface of the blood. Panel D shows all the samples 2 min after addition of the materials and after inversion of the tube. There was no evidence of clotting in the control tube. The high sphericity ORC aggregates produced a fully involved clot that adhered to the sample tube. The low sphericity ORC aggregates produced a less adherent clot, and the ORC fine fibers produced a modest clot. There was almost no clot formation detected in the tube treated with starch-based spheres.
Figure 4. Interaction of ORC powders with blood. Panel A is a side-view interaction of a high spherical ORC aggregate with blood. This artist’s rendering depicts the initial stages of the process in which nearly spherical ORC aggregates are thought to interact with whole blood. The aggregates penetrate into the blood and expand as their interfiber connections are loosened. During this process, some of the fibers also separate from the aggregates. Panel B is a side-view interaction of a low spherical ORC aggregate with blood, resulting in less penetration compared to a high spherical aggregate. Panel C is a side-view interaction of ORC fine fibers with blood, showing least penetration among the three ORC powders and most of ORC fine fibers interact with blood to form a layer clot to prevent the rest of materials from contacting blood.
aggregates variably penetrated the surface, depending on their sphericity, and dispersed beneath the interface into the larger volume of blood. This process is illustrated schematically in Figure 4. Once submerged, the ORC aggregates expanded as their interfiber connections were loosened. During this process, some of the aggregates also separated into fibers. The subsequent clotting reaction is consistent with that observed following exposure of blood to fabric ORC products, and is
appearing similar to the untreated control blood (Grade 0) in tube #1. When the tubes were inverted, only the high sphericity ORC aggregates appeared to produce a robust, adherent clot, as shown in Figure 3 panel D. In this qualitative in vitro model of clotting evaluation, there was a clear difference in the way the materials interacted with the blood. The starch-based spheres remained on the surface of the blood sample and appeared to float. In contrast, the ORC G
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 5. SEM images of ORC aggregates interacting with blood. This series of SEM images were obtained at brief intervals after the application of ORC aggregates to a blood sample as shown in Figure 3. Panel A (200×) shows the interaction of ORC aggregates at the initial blood-aggregate interface immediately after application. At this stage the aggregates are still relatively compact agglomerations of fine fibers and have been coated by a film of proteinaceous material and clotting factors (Please compare with Figure 1 for ORC fine fibers and aggregates in the absence of blood components). Panel B (250×) is a sample taken approximately 5 min later from the bottom of the tube containing the blood clot. At this stage the ORC aggregates have partially dispersed into ORC fine fibers and the fibers have been enveloped by a film of clotting components. The foreground shows a relatively intact aggregate particle, while the background reveals more dispersed fine fibers. Both the intact particle and the dispersed fibers are involved in the clot. Panel C (1500×) is a high magnification image of isolated fine fibers at 5 min showing their interaction with cellular components of the clotting reaction.
To further characterize the effect of sphericity on clotting, we produced ORC aggregates with sphericity values ranging from 0.51 to 0.79 with similar particle size distributions. In vitro clotting assays were performed on these materials as described earlier and were correlated with their sphericities. The results in Figure 7 indicate that the more spherical aggregates had greater
illustrated in representative scanning electron microscopy (SEM) images (Figure 5). The mechanism by which ORC promotes clot formation in vivo involved both local vasoconstriction as well as providing a matrix for platelet adhesion. As a result, the creation of the platelet plug, which will form the foundation of the fibrin clot, is accelerated.9,10 Red blood cells in contact with the ORC turn dark brown or black as the low pH causes degradation of the proteins and the formation of acid hematin.2 Clotting efficacy was quantified by comparing the mass of clotted blood remaining in the tube before and after inversion. Tubes were inverted, mechanically tapped 5 times with a tapped density analyzer, and allowed to rest for 2 min. Unclotted blood dripped out of the tube, and the remaining clotted residue in each tube was weighed; each material was tested in 6 replicates. The results are presented in Figure 6. The
Figure 7. In vitro clotting efficacy vs sphericity of ORC aggregates. Clotting efficacy, measured as the percent of the blood weight incorporated in the clot, was greatest for aggregate particles with the highest sphericity. Error bars are ± standard deviation (N = 4).
clotting efficacy than less spherical forms. At a sphericity of 0.79, clotting efficacy was nearly 96%, whereas at a sphericity of 0.51, the efficacy was less than 33%. Figure 4A, B illustrates the possible blood interaction processes of ORC aggregates (high sphericity of ORC aggregates and low sphericity of ORC aggregates). Comparisons of the surface area and surface energy data with the in vitro clotting results for the three different ORC powders (Figure 6) provide a framework for understanding their interactions with blood. The ORC fine fibers primarily floated on the surface of the blood with little penetration. The ORC low sphericity aggregates exhibited some penetration but not as deep as the high sphericity aggregates. The ability of the powders to penetrate into the blood appears to be directly related to the surface areas of the ORC materials. Higher surface area resulted in less effective penetration, whereas lower surface area materials sink more rapidly into the blood. Wettability is another distinguishing feature of these three materials. The ORC fine fibers and low sphericity aggregates
Figure 6. In vitro clotting performance. This in vitro clotting performance is determined by the percent of clotted blood (by weight) that was retained in the tube following topical application of test materials. The powder consisting of high sphericity ORC aggregates demonstrated the greatest clotting performance. Error bars are ± standard deviation (N = 6). *P < 0.01.
clotting efficacy for the high and low sphericity ORC aggregates was 95 and 38%, respectively. The clotting efficacy for ORC fine fibers and starch-based spheres was 26 and 19%, respectively. Untreated blood only retained 4% of its weight in the tubes. H
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 8. In vitro clotting/gelation performance. This in vitro clotting/gelation performance was performed on a total of six samples of recalcified citrated human blood, with test materials [ORC aggregate Sh(50) = 0.51, ORC aggregate Sh(50) = 0.76, ORC fine fibers or starch-based particles, respectively. This bench test for in vitro clotting/gelation study demonstrated that the average clotting time of recalcified citrated human blood, with each ORC aggregate [Sh(50) = 0.51 or Sh (50) = 0.76], was faster than the clotting time of recalcified citrated human blood with starch-based spheres or ORC fine fibers. The powder consisting of high sphericity ORC aggregates demonstrated the greatest clotting efficacy. Error bars are ± standard deviation. *P < 0.05.
because approximations were used in the calculations. However, these data suggest that starch-based spheres have less affinity for blood than the ORC materials and that the stronger cohesive forces in the starch-based spheres caused particle−particle interactions that result in the starch-based spheres agglomerating on the blood surface. Particles with both higher surface energies and higher surface areas to mass ratios (smaller particle size) have been found more difficult to disperse.13 The second in vitro clotting/gelation evaluation used an electromechanical clot detection method (viscosity-based detection system) to measure the clotting/gelation times for various weights of the test materials with recalcified citrated human blood. The results are displayed in Figure 8. The performance of the three various ORC powders as determined by this test exhibit the same trend observed for the first inversion test. In this case, the average gelation times are as follows: ORC aggregates [Sh(50) = 0.76] < ORC aggregates [Sh(50) = 0.51] < ORC fine fibers. Furthermore, both ORC aggregates are faster than the starch based particles. However, the ORC fine fibers have slower gelation times than the starch based spheres whereas in the inversion test their performance was similar. In the viscosity-based test, some of the ORC fine fibers remained on the surface throughout the duration of the test and were not involved in affecting the viscosity. The flotation was also observed for all the test materials in the inversion test upon the initial addition, but they then penetrated the surface to different degrees. One difference between the tests that may explain the variations in surface penetration is the weight of the test material per blood surface area. The values for the inversion test is 1.47 mg/mm2, whereas the weights per surface area for and the viscosity tests with 3, 5, and 7 mg are 0.0923, 0.154, and 0.215 mg/mm2, respectively. The different operating parameters of the two tests made it impossible to have overlapping weights/surface area. For the case of the fine fibers with a higher weight/surface area (1.47 mg/mm2), the larger amount of stacked powder provides additional force to the fibers to break through the surface. At
have slightly higher wettability values than that of high sphericity aggregates and as a result they are more hydrophilic. Powders with high surface areas and wettability values will interact with blood more rapidly than those with low surface areas and wettability values. Because the rate of gelation of ORC and blood is relatively fast, powders with higher surface areas and wettability are not able to integrate with blood and will remain near the surface. On the other hand, lower surface area powders with lower wettability are able to avoid gelation of blood at the surface and therefore penetrate into a larger volume of blood, resulting in more rapid and complete clot formation. The starch-based spheres have the highest surface area of all the materials and their degree of penetration into blood was minimal (Figure 3 and Table 1). In conjunction with the in vitro data (Figures 6 and 7 and Table 1), it appears that surface area is an important factor affecting penetration of a powder into blood. However, since ORC and the polysaccharide have different chemical structures, it was also critical to evaluate surface energetics to complete the comparison. The wettability of the starch-based spheres changes significantly with surface coverage (Figure 2D). At low coverages, wettability of the starch-based spheres is greater than wettability of ORC fine fibers and low sphericity ORC aggregates, but at higher coverages wettability is similar to that measured for high sphericity ORC aggregates. The dominant energy force that most clearly distinguished the starch-based spheres was the total surface free energy (Figure 2A and Table 2), which was much greater than all of the three ORC powders. Higher surface energies are associated with stronger cohesive forces and particle−particle interactions. This high total free energy is directly related to a high work of cohesion and a relatively small Wad/Wcoh ratio. As shown in Table 2, the Wad/Wcoh ratios for starch-based spheres, ORC fine fibers, ORC aggregates Sh(50) = 0.51, and ORC aggregates Sh(50) = 0.76, are 0.792, 1.02, 0.982, and 1.08, respectively. The variations in ORC fine fiber and ORC aggregate Wad/Wcoh ratios are too small to draw any meaningful conclusions about the differences in their behaviors I
DOI: 10.1021/acsbiomaterials.7b00382 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 9. Bactericidal efficacy of ORC aggregates. The test organisms were as follows: (1) Staphylococcus aureus (MRSA) ATCC 33591; (2) Staphylococcus epidermidis (MRSE) ATCC 51625; (3) Streptococcus pneumoniae (PRSP) OC 21411; (4) Enterococcus faecium (VRE) ATCC 700221; (5) Bacillus subtilis ATCC 6633; (6) Bacteroides f ragilis ATCC 25285; (7) Branhamella catarrhalis ATCC 25238; (8) Clostridium perfringens ATCC 3624; (9) Clostridium tetani ATCC 19406; (10) Corynebacterium xerosis ATCC 373; (11) Enterococcus cloacae ATCC 13047; (12) Enterococcus faecalis ATCC 29212; (13) Escherichia coli ATCC 8739; (14) Klebsiella pneumoniae ATCC 10031; (15) Micrococcus luteus ATCC 4698; (16) Mycobacterium phlei ATCC 11758; (17) Proteus mirabilis ATCC 12453; (18) Proteus vulgaris ATCC 13315; (19) Pseudomonas aeruginosa ATCC 9027; (20) Pseudomonas stutzeri ATCC 17588; (21) Salmonella enteritidis ATCC 13076; (22) Serratia marcescens ATCC 13880; (23) Shigella sonnei ATCC 9290; (24) Staphylococcus aureus ATCC 6538; (25) Staphylococcus epidermidis ATCC 14990; (26) Streptococcus agalactiae ATCC 624; (27) Streptococcus pyogenes ATCC 12384; (28) Streptococcus salivarius ATCC 13419; (29) Lactobacillus rhamnosus ATCC 7469. See the raw data in Table S2.
were done in different swine and rat animal bleeding models to characterize the hemostatic performance of ORC aggregates of high sphericity, and this type of delayed rebleeding was not detected in those studies either. 3.5. In Vitro Bactericidal Activity. To further characterize the biological activity of high sphericity, ORC aggregates as they relate to other topical absorbable hemostats, we examined the bactericidal properties of the ORC aggregates and six currently marketed products. We first tested the ORC aggregates against a panel of 29 bacteria that were used to establish the antibacterial activity of marketed ORC fabric products. There was a 3 to 4 log reduction in viable organisms for all 29 bacterial strains tested at 2.5% w/v, as shown in Figure 9. The inoculum was on the order of 1 × 104 to 1 × 105 organisms; after incubation with ORC aggregates the number of organisms recovered was below the detection limit (
