Cuttlebone Fillers for Topical Hemorrhage and Wound Healing: In Vitro Insights and a Suppository Model


 BackgroundWhile the treatment of numerous anorectal disorders remains challenging, this paper aims to add an impact and suggests a product prototype with marine-derived non-toxic cuttlebone fillers for clinical use. MethodsElemental composition, hemostatic and antibacterial potential of cuttlebone fillers were tested in vitro while comparing with a number of clotting powders. Witepsol-based suppositories with lidocaine hydrochloride and cuttlebone fillers were further developed. ResultsCuttlebone microparticles (CB-1) and modified alkali-treated cuttlebone microparticles (CB-2) were analyzed in terms of hemostatic efficiency and as fillers for rectal suppositories for the first time (Fig. 1). Bioinorganic elements of cuttlebone, such as iron, zinc, copper, calcium and magnesium were found to be supportive for wound healing. Modified, chitosan-enriched cuttlebone filler showed decrease in clotting time by 20%. Cuttlebone fillers demonstrated no antimicrobial activity against S. aureus and P. aeruginosa. Suppositories with cuttlebone fillers have demonstrated favorable characteristics, such as melting point in a 36.0–37.0 °C temperature range, dissolution time no longer than 30 min and gradual release of the anesthetic drug. ConclusionBased on the primal, though essential to conduct in vitro test results, application of cuttlebone fillers could open a new page in the development of successful naturally-based hemostatic and wound healing products.


Abstract
Background While the treatment of numerous anorectal disorders remains challenging, this paper aims to add an impact and suggests a product prototype with marine-derived non-toxic cuttlebone llers for clinical use.

Methods
Elemental composition, hemostatic and antibacterial potential of cuttlebone llers were tested in vitro while comparing with a number of clotting powders. Witepsol-based suppositories with lidocaine hydrochloride and cuttlebone llers were further developed.

Results
Cuttlebone microparticles (CB-1) and modi ed alkali-treated cuttlebone microparticles (CB-2) were analyzed in terms of hemostatic e ciency and as llers for rectal suppositories for the rst time ( Fig. 1).
Bioinorganic elements of cuttlebone, such as iron, zinc, copper, calcium and magnesium were found to be

Background
According to traditional practices of Chinese and Indian medicines, cuttlebone has been used to treat various ailments 1,2 . Regrettably, nowadays previously attained knowledge is left out of consideration.
Contemporary research is mostly focused on the use of cuttlebone for bone tissue engineering [3][4][5][6][7][8][9] , while its wound healing potential is rarely mentioned. Jang and co-authors 10 were the pioneer researchers who described the effect of cuttlebone chitin-based extract for wound healing. In their study, increased reepithelization of rats' burn injured skin and reduced expression of pro-in ammatory cytokines were observed.
Cuttlebone is an internal skeleton of marine mollusk Sepia o cinalis, class Cephalopoda 11 . Main constituents of cuttlebone are aragonite -a polymorph of calcium carbonate, polysaccharide β-chitin and trace minerals 12 . In average, the amount of chitin in cuttlebone is too low to use it as an effective source material for chitosan production as compared with other crustaceans and cephalopods like shrimp, crab, crill, lobster or squid. Those are rich in chitin and therefore are cost-effective to use for the preparation of commercially employable chitosan-based materials for biomedical and pharmaceutical applications, food industry or wastewater treatment.
Within the last decade, international patents submitted by Chinese researchers disclosed a number of recipes from Chinese medical heritage where cuttlebone is an ingredient in the composition of healing mixtures, ointments and pills. Cuttlebone has been mentioned in Chinese traditional medicine for bleeding control 13 . Empirical practice of nowadays sailors has shown promising results of cuttlebone for epidermal wound healing and bleeding control. Usually, on a boat, fresh cuttlebones were dried in direct sunlight for several days, then crushed and powdered, and later applied to skin wounds when necessary.
Continuing the empirical practice, cuttlebone was used for wound healing by sailors and their families for decades [1].
Despite the fact that powder is considered as one of the simplest pharmaceutical dosage form to prepare, it could serve as a basis for further developments. For instance, powdered materials are used for making solid dosage forms like effervescent tablets, tablets, granules, capsules or dry-powder inhaler; liquid dosage forms like suspensions; and semi-solid dosage forms like suppositories, suspended gels, creams or liniments.
The retrospective analysis of Medicare bene ciaries (2018) identi ed that more than eight million people had wounds with or without infections 14 . Bleeding is the most common symptom in grades 1 and 2 hemorrhoids, while infection in a perianal area may present as different types of abscesses, perineal sepsis, stulae which commonly occur in patients with acute leukemia 15,16 . Suppositories containing local anesthetics agents, steroids, astringents, vasoconstrictors, protectants, antiseptics, keratolytics, veno-tonics, coagulants of necrotic tissues, analgesics, herbal (rhubarb, aloe) 17 and animal (shark liver oil) components are known to be used for the treatment of anal disorders. While the treatment of numerous anorectal disorders remains challenging, this paper aims to add an impact and suggests a product prototype for clinical care.
This study aims to investigate cuttlebone materials suitable for topical application in wound healing and bleeding control, as well as to develop Witepsol-based suppositories for anorectal disorders with cuttlebone llers.

Aim
This study aims to investigate cuttlebone materials suitable for topical application in wound healing and bleeding control, as well as to develop Witepsol-based suppositories for anorectal disorders with cuttlebone llers.

Design
To deepen the topic, following objectives were achieved: i) modi cation of the original cuttlebone material by means of alkaline deacetylation; ii) characterization of both original and modi ed materials by means of elemental composition and antibacterial potency, iii) testing of cuttlebone materials in vitro in comparison to other powders/granules as for a hemostatic effect; and iv) subsequent development and testing of the suppositories with two cuttlebone llers.
Commercial hemostatic chitosan-based product Celox [1]; bentonite as a prototype of a clay-based hemostatic agent; talc as a negative control 18  Electronic laboratory notebook platform was not used. Summary graphic illustration was created in BioRender.com.

Preparation of cuttlebone materials
Whole cuttlebones, including dorsal and lamellar parts, were crushed and milled in an agar mortar; the fraction (particle size 32-45 μm) was collected using sieves. Two types of cuttlebone powders were used in this study: 1) untreated cuttlebone microparticles (CB-1), and 2) cuttlebone microparticles treated with 40% NaOH solution at 80.0 °C for 6 h under continuous stirring (CB-2). After alkaline treatment, the material was thoroughly washed with cooled decarbonized distilled water until neutral pH and dried in a thermostat (Binder ED series 53, Binder GmbH, USA).

X-ray uorescence analysis
The elemental composition of powdered samples was evaluated by X-ray uorescence (XRF) spectrometric analysis (Bruker X-ray S8 Tiger WD, Bruker AXS GmbH, Karlsruhe, Germany) and employing Spectraplus quant-express method.

Microbiological assay
The concentrations of bacteria cultures were adjusted by comparing them to the concentrations in standard tubes with McFarland turbidity of 0.5 (1.5×108 CFU). Muller-Hinton agar powder was used as a culture medium. The inhibiting activity was tested by the disc diffusion method and in two different ways. First method for testing antibacterial activity was adapted from Immanuel et al. 19 where antimicrobial activity of a marine shell powder was tested and second method where the suspension from demineralized cuttlebone powder was tested. For the rst method, suspensions of cuttlebone powders were prepared in 2%, 4% and 6% (w/v) concentrations by using sterilized distilled water. Sterile paper discs (6 mm diameter) were impregnated with the respective suspensions for 5 days in sterile glass jars that were kept at 25±0.1°C temperature in an incubator shaker (KS 4000 i control, Werke GmbH & Co. KG, Germany) at 80 rpm. Further, the discs were placed in an agar medium by using a sterilized forceps. For the second method, suspensions from demineralized cuttlebone powder were prepared by adding 5% lactic acid. The amount and concentration of lactic acid for demineralization of CaCO 3 in cuttlebone material was calculated according to the Equation 1 and maintained at pH=6.5: The obtained opaque solution was stirred at 50 rpm while using a magnetic stirrer for 2 h. Wells in an agar medium were lled with the solutions by using a micropipette. For both methods, the zone of inhibition was observed after the incubation of Petri dishes at 37.0 °C temperature for 24 h. Four samples were tested simultaneously for each sample concentration and bacterial strain.

Exothermic reaction test in vitro
Exothermic reaction was tested in vitro according to Li et al. 20 . Five (5) g of sample powder were placed into a plastic tube and 5 mL of distilled water were added. The temperature reading was recorded before each testing and after 1 min when water was added to the powdered material. Mercury thermometer (Labothermometer, Amarell GmbH&Co., Kreuzwertheim, Germany) with a measuring range from 0.0 to +30.0 ±0.1 °C was inserted into a 25 mL plastic tube. All tests were made in triplicate and the average meaning ± standard errors of deviation (SED) were recorded as results.

Water contact angle
Powdered samples (0.3 g) were pressed into a pellet using a tablet press machine. Contact angles were measured using Theta Light optical tensiometer (Biolin Scienti c, Sweden). Data for each sample type (n=5) was recorded within 10 sec and the mean values of left and right contact angles were obtained.
The results are expressed as an average of mean contact angle of 5 measurements estimated within 10 sec ± SED.

Coagulation assay
Coagulation assay was performed by testing blood clotting capacity and blood coagulation rate. Blood clotting capacity was tested as described by Liu et al. 21 with some modi cations. Brie y, 200 μL of citrated blood were added to 10 ±0.2 mg of each powdered sample in a 6-well plate. To initiate blood clotting, 100 μL of 0.025M CaCl 2 solutions were then added and samples were incubated in a thermostat at 37.0 °C for 10 min. After incubation, 15 mL of distilled water were carefully added to each sample without disrupting a formed clot. Ten (10) mL of the resulting hemoglobin solutions from each well were taken and centrifuged at 1000 rpm for 1 min, followed by incubation of the supernatant at 37.0 °C for 1 h. The absorbance of the resultant solutions was measured at 540 nm by using a spectrophotometer (Varian Cary 50 UV-VIS, Varian Inc., Netherlands). The absorbance of blood (200 μL) in 15 mL of distilled water (without any powders added) was used as a reference value. For the quantitative evaluation of clotting ability, the absorbance of hemoglobin from an aqueous solution was taken into consideration, while hemoglobin entrapped in the samples was not counted. All measurements were carried out in triplicate and the average meaning ± standard errors of deviation (SED) were recorded as a result.
Blood coagulation rate was measured imitating the Westergren method and a dynamic rheological experiment as described by Periayah 22 . Blood coagulation rate was measured by observation of clot formation (aggregation of red blood cells, RBCs) during inversion of Eppendorf tubes within time points.
For testing of blood coagulation rate, 0.003 ±0.0004 g of a measured powdered sample was poured directly into 1.5 mL Eppendorf tube. Citrated blood was incubated at 37.0 °C for 30 min. After 30 min, 200 μL of blood sample was added to each Eppendorf tube with a powdered sample and coagulation was initiated by adding 100 μL 0.025M CaCl 2 solution. Blood coagulation time was recorded every 30 sec, and after 10 min the test was stopped. Citrated blood mixed with a CaCl 2 solution was used as a control.
Each type of sample was tested in triplicate simultaneously. The results are expressed as an average mean of 3 measurements ± SED.

Visualization
Clots obtained after clotting test were washed with distilled water, frozen and lyophilized (Christ Alpha 2-4 LSC, Osterode am Harz, Germany). Scanning Electron Microscopy (SEM) (FEI Quanta 200 FEG, Oregon, USA) was used to obtain microphotographs of the clot surface. Summary graphic illustration (Fig. 1) is created with BioRender.com

Characterization of suppositories with cuttlebone llers
The open capillary tube method was used to nd the melting point according to the description in the European Pharmacopeia. Suppository sample was placed in a cylinder and the temperature of water in the environmental medium was raised by 1°C temperature per min. A temperature point of the rst drop of the melted base was recorded. For density determination, a glass cylinder was lled with 10 mL of distilled water. A suppository sample (2.0 g) was immersed into water, and the reading of the increased volume is recorded and the density was calculated.
For the determination of dissolution time, a cylindrical glass with distilled water was put into a water bath (37.0±0.1 °C), and temperature inside a glass is equilibrated. A suppository sample was immersed into a cylindrical glass and trapped slightly to avoid otation. The content of a cylindrical glass was stirred at 120 rpm until the dissolution of the sample. A time span no longer than 30 min was considered as optimal.
Release of lidocaine hydrochloride was performed by using the basket method (Erweka DZT, Erweka GmbH, Germany) according to the European Pharmacopeia. The basket was rotated at 120 rpm in 500 mL phosphate buffer saline (PBS) (pH=6.8, 37.0±0.5 °C). At predetermined time points, aliquots of 5 mL were withdrawn and ltered, while the volume of the dissolution uid in a dissolution apparatus ask was compensated immediately by adding the same volume of PBS. The recording of drug concentration in aliquot was measured spectrophotometrically at 265 nm and calculated by using the standard curve (0.5, 0.25, 0.0125 and 0.0625 mg/mL; R 2 =0.9925). Data was expressed as the mean value of 3 tests ± SED.

Characteristics of cuttlebone materials
Two types of cuttlebone materials were prepared and analyzed: CB-1 and CB-2 powders. In this work, CB-1 is assumed as a "minerals/chitin" complex, and thus reveals original characteristics of the material. CB-2 powder is a new modi cation and could be assumed as a "minerals/chitosan" complex ( Fig. 2).
According to the results of the elemental analysis (Table 1), zinc, magnesium, potassium, iron and copper were found in both cuttlebone materials. In contrast to bentonite, no aluminum was found in cuttlebone samples [1]. Oppositely to the expectations, all the samples showed no antibacterial activity against the selected bacteria strains. Zones of bacterial growth inhibition for cuttlebone materials were not observed for S. aureus and P. aeruginosa.
Cuttlebone materials in terms of hemostatic potency All tested materials showed temperature increase while conducting the exothermic reaction test, however values varied depending on a composition. Minimum temperature increase was observed for CB-1 (0.1±0.1°C), and noticeably higher temperature increase, but within warrantable limits, was observed for CB-2 (1.1±0.2°C), Celox (1.3±0.3°C) and bentonite (1.3±0.2°C). Temperature increase for calcium carbonate and talc were 0.2±0.1°C and 0.3±0.2°C, respectively.
All values of hemoglobin absorption were signi cantly low if compared with the control sample (Fig. 3). Herein, lower absorbance value indicates a higher capacity of a sample to entrap hemoglobin and more likely to initiate clot formation from the perspective of protein (hemoglobin) adsorption 21,23 . As

SEM microphotographs of clots with cuttlebone
According to macroscopic observation all samples formed clots within 20-30 min of incubation. Fibrin matrix formation, recognizable as a threadlike mesh, was clearly visible on the surface of both cuttlebone materials and Celox (Fig. 4). Mostly oval-shaped erythrocytes were visible on the surface of cuttlebone samples, indicating non-disturbance of RBCs morphology during contact with cuttlebone microparticles (Fig. 5). Structured aggregates of RBC on CB-1, CB-2 and Celox were visible (Fig. 5a-c). Formation of erythrocyte aggregates was most likely induced by chitosan fragments and, as a result, additional brin links were formed. Single erythrocytes were visible on a surface of bentonite, talc and calcium carbonate ( Fig. 5d-f). Distorted plate aggregates were visible for talc and calcium carbonate (Fig. 5e, f).

Suppositories with cuttlebone llers
Suppositories with cuttlebone powdered materials CB-1 and CB-2 were prepared by pour molding technology and choosing Witepsol H35 (W-H35) as a base: W-H35/CB-1 and W-H35/CB-2. Both formulations showed melting points agreeable to the European Pharmacopeia recommendations: 36.2±0.1 °C and 36.3±0.1 °C, respectively. The average weight of prepared suppositories was found not to exceed the established limits for suppositories as single-dose preparations: 1.85±0.1 g and 1.86±0.1 g, respectively. Density (ρ) was measured as 0.16±0.01 g/cm 3 and 0.11±0.04 g/cm 3 , respectively. According to the European Pharmacopeia, dissolution time for suppositories in the physiological environment (37.0 ) needs to be less than 30 min. Both suppository formulations satis ed the requirement: 26±2 min and 27±1 min, respectively.
Gradual release of lidocaine hydrochloride into dissolution medium was observed within 40 min while showing in vitro early onset of drug activity and the appropriate drug-release pro le (Fig. 6). Drug release pro les of prepared cuttlebone-lled suppositories were found to be similar to the drug release of a commercial suppository (Doloproct). Lidocaine hydrochloride release into dissolution medium was the lowest for W-H35/CB-2.

Discussion
Modi cation of the original cuttlebone material via alkaline deacetylation allows to convert inward chitin into chitosan -a well-known clotting agent and wound healer of a marine origin. Alkaline deacetylation is a conventual procedure for chitosan preparation 25 form crustacean waste, while skipping the demineralization step in the same procedure for cuttlebone allow to retain its bioinorganic elements and aragonite. This gives a double advantage because bioinorganic elements of cuttlebone are bene cial for wound healing while a dominant amount of aragonite creates a mechanical barrier for bleeding site (will be discussed in details later).
Chitin and its derivative chitosan are both linear co-polymers consisting of 2-acetamide-2-deoxy-β-Dglucopyranose and 2-amino-2-deoxy-β-D-glucopyranose, which are connected by β(1→4) linkage. The characteristics of chitin, as in CB-1, for biomedical applications have been thoroughly studied. Chitin is a non-toxic and biodegradable natural polymer, used as a matrix in tissue engineering enhancing the surrounding tissue ingrowth and avoiding scar formation. Chitin membranes have shown antimicrobial and broblast growth activity. Chitin monomer N-acetylglucosamine controls collagen synthesis and improves granulation thus facilitates wound healing 26,27 . Chitosan (as in CB-2) is known for antimicrobial activity, good biocompatibility, biodegradability, non-toxicity and solubility in acidic aqueous solutions.
So far, scant investigation on the cuttlebone antimicrobial activity has been performed. However, despite the favorable composition which includes Zn, Cu, chitin and/or chitosan and more so, despite the results of other studies showing antimicrobial activity of cuttlebone -in our study the fact was not proven. Antimicrobial activity of CB-1 and CB-2 powders was tested in two ways by using: 1) paper disc diffusion method and 2) fully demineralized cuttlebone materials while imitating normal skin pH in a 4.5-6.5 range. In order to emphasize the pH in uence, the second method was performed at pH=6.5 because lower pH could affect the results to positive values due to pH-sensitivity of bacteria itself, but not because of the antimicrobial potency of the samples. Strains of bacteria found on the skin surface, such as Staphylococcus aureus and Pseudomona aeruginosa, were chosen for the study.
A few theoretical models have been proposed for the explanation of the antimicrobial activity of chitosan. One of them is explained by the electrostatic interaction occurring between the negatively charged zones of the bacterial membrane and the protonated NH 3+ in the acidic medium; NH 3+ groups block the connections of the membrane with Ca 2+ and thus determine the death of pathogenic cells. Free amino groups that are present in the chitosan structure determine the antimicrobial activity in aqueous solutions below their pKa value (6.0<pH>6.50). Another proposed mechanism explains that chitosan binds with microbial DNA, and the chelation of metals, suppressing the growth of a bacterial cell, occurs 28,29 .
The antibacterial action of zinc and copper against Staphylococcus aureus, Escherichia coli and Bacillus has been already proven 30 . Even though the presence of Zn and Cu and the antimicrobial potency of charged chitosan in cuttlebone composition at pH=6.5 was anticipated, the evaluation of antimicrobial activity showed neither bactericidal nor bacteriostatic activity. Concentration of Zn in the cuttlebone composition (Table 1) is negligible, thus it probably could not manifest positively for antimicrobial activity as in the study by Beherei et al 31 . Interestingly if for bone tissue studies, Dogan and Okumus 3 revealed that cuttlebone "is associated with decreased formation of free radicals in soft tissue, and it allows bone healing without causing oxidative stress." In their study, according to the data on biochemical and histological parameters, no in ammatory or foreign body cells were observed and did not cause (re)infection after implantation of non-modi ed cuttlebone block into a bone defect surrounded by soft tissue.
Other studies focus on the polysaccharides extraction from cuttlebone using methanol and ethylenediaminetetraacetic acid -both potential antimicrobic agents [32][33][34] . Therefore, successful results on antimicrobial activity of polysaccharide extracts from cuttlebone have been achieved against grampositive and gram-negative pathogenic bacteria and fungi.
Elemental composition of commercially available cuttlebone, CB-1 in Table 1, is similar to the results for cuttlebone from different coastal zones where biomedical safety aspects were also discussed 4 . Components of cuttlebone have a favorable impact on wound healing. For instance, zinc is an important trace element for a number of living organisms, because it is a structural component of proteins which positively affects their functions. It appears to be one of abundant transition metals in blood composition, binding plasma proteins and modulating their structure and functions by responding to a dynamic microenvironment. By this, zinc could be considered a relevant mediator of hemostasis and thrombosis 35 . Studies have shown that zinc cations are an important cofactor in Factor XII contact activation. Therefore, zinc particles may have an impact on a coagulation process. Magnesium suppresses skin in ammation, while a duet of magnesium and calcium in uences cell proliferation and differentiation. Iron de ciency is associated with deceleration in wound healing. Copper modulates melanin synthesis and stimulates maturation of skin collagen. Trace amount of copper in cuttlebone composition is related to the protein hemocyanin which is essential for respiration of cuttle sh  .
Elemental composition of clays, like bentonite, commonly includes aluminum. Aluminum was found in talc, zeolite 21 and QuikClot 21 (Table 1). Aluminum is known as a suppressant of collagen synthesis, especially if calcium and magnesium are de cient 37 . This fact could be the main cause for negative impact of aluminum-containing materials when they come into contact with injured skin, including heat generation and problematic wound healing.
Medicine constantly refers to pros et contras for the use of naturally-based materials in bleeding control. Most dramatic adverse effects of naturally-derived hemostatic agents are in-contact exothermic reaction, insu cient biocompatibility and di culties of agent removal after a surgery. Heat generation, as a result of an exothermic reaction, is the most frequently encountered adverse effect of aluminosilicates (QuikClot and Combat Gause). Fibrin-based hemostatic agents ( brin sealants, Tisseel; platelet gels, Vitagel) are known for the transmission of infectious diseases that are common in biological materials.
Clay-based products (bentonite, kaolin and smectite products, such as WoundStat 24, 38 ) may be a cause of distal thrombosis due to occlusion of arterial ow. Di culties in removal of post-reacted portions of a material have sometimes been resulted when using mineral-based products 39 .
"Test-tube" experiments prior to cell-culture and in vivo tests are important when new prototypes are rst introduced. As was stressed by Wiegand et al. 40 , "the application of controlled in vitro techniques might serve as a screening tool in the development of new hemostatic agents." Nowadays, worldwide research is concentrated on comparability between results of in vitro and in vivo tests 41,42 . In contrast to conventional belief that in vitro tests should be a "mirror re ection" to animal models, true value of forehead in vitro testing is to complement a whole picture and bene t from the interpretation of the results in a multidisciplinary format.
So-called "exothermic reaction" is an undesirable result from a contact between a hemostatic agent and a bleeding site. However, it is a quite often, complex physical-chemical-biological reaction of a living organism. It can cause pain, discomfort, tissue burns, necrosis, and therefore aggravates or severely disrupts proper wound healing. Elemental composition of a hemostatic agent determines a degree of an exothermic reaction, because speci c elemental components, mainly aluminum, could cause excessive and accelerated absorption of water at a bleeding site. Subsequently, heat is generated as a result of anomalous accelerated formation of a clot 43 . The reaction originates from rapid dehydration of aqueous blood components followed by an increased local concentration of clotting factors. As a result, skin burns or even tissue necrosis could occur.
Therefore, hemostatic materials showing in-contact low temperature increase are highly desirable.
Necrosis, as a worst scenario, was observed during the in vivo study by Li et al. 44 . In their study, the temperature increase of zeolite granules and QuikClot was 6.9±0.4°C and 44.6±1.0°C, respectively. The authors explained the mechanism of heat generation by excess of Ca 2+ ions (11.4%) in a composition of QuikClot. In comparison to our study, the amount of calcium in cuttlebone powders was ve-fold higher: 50.7% and 50.9% for CB-1 and CB-2 powders, respectively (Table 1). However, temperature increase for cuttlebone materials was negligible: 0.1±0.1°C and 1.1±0.2°C, respectively. Calcium in cuttlebone composition is in an aragonite form (i.e. calcium carbonate -a calcium salt with low solubility in water), thus only a negligible portion of ionized calcium in aqueous medium could appear in a short period of time, time period which is necessary for clot formation. Moreover, following the principle of a coagulation cascade, the threshold level of Ca 2+ is essential for clotting to start 24 . By taking into consideration all the above-mentioned facts, excess heat generation of QuikClot 44 should be explained by other reasons, most likely due to aluminum in its composition.
In terms of hemostatic properties, CB-2 clotting time was shorter by 20.5% if compared with a control sample. The value correlates well with data for chitosan and some commercial products, such as Gelfoam, Surgicel and dehydrated QuikClot 44 .
Liu et al. 21 analyzed blood clotting ability of halloysite nanotubes. In their study, the absorption value of halloysite, a natural inorganic material, was similar to a control sample, demonstrating poor ability to affect blood clotting. However, it needs to be made apparent that blood clotting ability test based on hemoglobin absorbance measurement is more relevant for sponges, ber mats and other 3D porous materials, because it shows capacity of a sample to absorb blood by volume. Generally, higher absorptive values are found for porous materials as compared with powdered materials [45][46][47] and that was the case in this study too.
One should appreciate the dual nature of blood: hydrophilic and hydrophobic characteristics in regards to water content and formed elements of blood, respectively. Hydrophilicity of a material is usually characterized by measuring its water contact angle. A hydrophilic surface of a material promotes coagulation cascade, because human blood as a hydrophilic substance (contains approximately 83% of water) and could permeate into a hydrophilic material much easier and faster 48 . Thus, in terms of surface wettability, clotting agents with lower contact angle (hydrophilic) are advantageous. However, only moderate surface hydrophilicity could be deemed as appropriate. For example, in this study contact angles of calcium carbonate and talc are too low (15.6±2.5° and 14.8±0.8°, respectively) to accept them as suitable ones for hemostatic applications.
On the other hand, vast research has been dedicated for studying blood coagulation at biomaterial interface, where blood coagulation and platelet adhesion are examined as a main downside to using implantable medical devices. Herein, hydrophobicity is a determining factor of protein adsorption due to surface chemistry and charge -more proteins will adsorb to hydrophobic surfaces. Blood proteins, such as albumin, brinogen and Factor XII, are more adherent onto hydrophobic surfaces and therefore mediate platelet adhesion and thrombus formation. Factors such as contact time, topography and roughness, surface free energy and/or functional groups are the characteristics of a material which could also affect an eventual result 23,49 .
Chemical and physical characteristics of surfaces induce the dynamics of blood protein adsorption onto an arti cial surface. Hence, plasma proteins by themselves initiate subsequent clot formation by modulating a number of reactions 23 . Powdered material could enhance clot formation by absorption of blood uids, acts cohesively and adhesively, thus accelerating agglomeration of cells and stopping hemorrhage by clot formation 18,50 . Similarly, cuttlebone while crushed into a powder, has an ability to form a mechanical barrier.
In summary, presence of aluminum in some clay-based or aluminosilicate hemostatic agents, in parallel with their de ciency in calcium and magnesium, obviously argues against proper wound healing. Both cuttlebone materials are rich in various bioinorganic elements supportive for haemorrhagic control and wound healing, are most likely biocompatible with injured skin and could be described as hemostatic agents with an ability to form a mechanical barrier.
Witepsol is a typical industrial suppository base consisting of a mixture of mono-, di-and triglycerides. Witepsol is easy to handle; its melting procedure is facilitated by the non-overheating characteristic of the base. For this reason, Witepsol bases are widely used on the industrial scale for the preparation of pharmaceutical suppositories. Lidocaine hydrochloride, as an anesthetic, is commonly used in preparations such as suppositories, creams, gels and solutions. Solid para n (adjuvant) is commonly effective as a hardener and for the rise of the melting point of fatty base formulations. Carbopol (adjuvant) is a mucoadhesive material which is helpful for the enhancement of drug dissolution in biological environment.
Melting points of both formulations were in the 36.0-37.0° C temperature range; as this characteristic is critical for a suppository under physiological conditions. Mass uniformity is an important characteristic of single-dose preparations because it ensures (a) equal distribution of active and adjuvant ingredients and (b) the therapeutic window of a drug (the therapeutic window (or the pharmaceutical window) of a drug is the range of drug dosages which can treat a disease effectively without having toxic effects, author's note). The deviation (%) of suppositories weight was ±0.1, whereas the required deviation could be up to ±5% for n=20. Preparation of suppositories by molding is generally accepted as an effective and time-saving method in the pharmaceutical industry.
Drug release was performed in PBS at pH=6.8 at 37 °C with the aim to simulate the physiological pH of a rectum. As release of lidocaine hydrochloride into dissolution medium was slower for W-H35/CB-2 -a chitosan-enriched cuttlebone material -it proves the existing fact that chitosan has an impact on a controlled drug release. Other studies show the e ciency of chitosan as a drug reservoir, demonstrating the functionality of a chitosan/lidocaine system for the achievement of a prolonged anesthetic effect 51 .
On the other hand, the drug release pro le of rectal preparations does not fully re ect rectal absorption, and, therefore, the drug bioavailability. There are several physical-chemical factors affecting the rectal absorption, such as the drug solubility in a vehicle, the particle size, the nature of the base, the spreading capacity and other related physical factors. The release rate of lidocaine hydrochloride into the rectal uid was expected to be high because its solubility in water is 50 mg/mL, while the pure drug is highly hydrophobic 52 .
To conclude, chemical characterization of any hemostatic agents should be of exorbitant concern, because biocompatibility, especially hemocompatibility, is an essential measure to predict fortunate of any newly tested material. Elemental composition of any clotting agent is crucial despite any other characteristics, because it could arguably have an immediate impact on living tissue and further degree of success in wound healing. Cuttlebone llers are characterized as multicomponent materials with positive impact for bleeding control and wound healing. Cuttlebone llers are supposed to be applicable to treat topical wounds directly or as a llers for Witepsol-based suppositories to treat anorectal disorders with bleeding symptoms.

Conclusions
Bioinorganic elements of the cuttlebone, such as zinc, iron, calcium, magnesium and copper, are supportive for wound healing. Cuttlebone is aluminum-free, and therefore has an advancement over aluminosilicate hemostatic minerals as aluminum is associated with suppressed collagen synthesis.