Modification 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 preparation25 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 beneficial 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-D-glucopyranose 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 fibroblast 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 influence, 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 NH3+ in the acidic medium; NH3+ groups block the connections of the membrane with Ca2+ 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, occurs28, 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 inflammatory or foreign body cells were observed and did not cause (re)infection after implantation of non-modified 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 32-34. Therefore, successful results on antimicrobial activity of polysaccharide extracts from cuttlebone have been achieved against gram-positive 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 inflammation, while a duet of magnesium and calcium influences cell proliferation and differentiation. Iron deficiency 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 cuttlefish 36–30.
Elemental composition of clays, like bentonite, commonly includes aluminum. Aluminum was found in talc, zeolite 21 and QuikClot21 (Table 1). Aluminum is known as a suppressant of collagen synthesis, especially if calcium and magnesium are deficient 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, insufficient biocompatibility and difficulties 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 (fibrin 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 WoundStat24, 38) may be a cause of distal thrombosis due to occlusion of arterial flow. Difficulties 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 first 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 reflection” to animal models, true value of forehead in vitro testing is to complement a whole picture and benefit 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 specific 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 Ca2+ ions (11.4%) in a composition of QuikClot. In comparison to our study, the amount of calcium in cuttlebone powders was five-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 Ca2+ is essential for clotting to start 24. By taking into consideration all the above-mentioned facts, excess heat generation of QuikClot44 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, fiber 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 45-47 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, fibrinogen 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 artificial 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 fluids, 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 deficiency 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 paraffin (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 efficiency 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 profile of rectal preparations does not fully reflect 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 fluid 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 fillers are characterized as multicomponent materials with positive impact for bleeding control and wound healing. Cuttlebone fillers are supposed to be applicable to treat topical wounds directly or as a fillers for Witepsol-based suppositories to treat anorectal disorders with bleeding symptoms.