Tania Hossain, scientist, at Center for Advanced Research in Sciences (CARS), for allowing me to use their facilities and share their thoughts. Furthermore, I would like to thank all professors and staff of the Department of Chemical Engineering, BUET.
Background
Fibrin glue is one of the most widely used commercial bioadhesives made from blood plasma. In the first stages of development and application trials, human plasma and bovine thrombin were used to make fibrin glue.
Objectives and Outcomes of the Study
Characterization of different biopolymer-supported autologous adhesive in terms of adhesive strength, setting time, cytotoxicity and morphological changes by varying the composition of the adhesive. The effect of the biopolymers on the adhesive strength as well as the optimal compositions of components in the autologous glue will be determined.
Organization of the Thesis
4 glue and finally the importance of the development of autologous fibrin glue which highlights the necessity of this study. Finally, morphological analysis of the fibrin glue is also discussed based on the SEM and EDS data.
History and Origin of Fibrin Adhesives
There is a large number of research papers on the history, development and applications of fibrin glue. It also discusses the various components of fibrin glue and its effect, the working principle of fibrin glues and the manufacturing process of commercial fibrin glues.
Types of Adhesives Used in Surgical Applications
Hemostatic Agents
Some of the commercially available hemostats are porcine gelatin (such as Gelfoam and Surgifoam), bovine collagen (such as Helitene, Avitine, and Instat), and oxidized regenerated cellulose (such as Surgicel) (William D. Spotnitz 2007). For example, thrombin interacts with fibrinogen in the patient's blood to accelerate the formation of a fibrin clot.
Tissue Adhesives and Sealants
Different mechanisms of adhesion are briefly discussed in the next section, which can provide a better understanding of the functional mechanisms of bioadhesives and their performance and facilitate innovations in new and innovative designs.
Mechanisms and Theory of Adhesives and Adhesion
- Mechanical interlocking
- Intermolecular bonding
- Chain entanglement
- Electrostatic bonding
It is believed that electrostatic forces are induced by these charges, which may play a role in the intrinsic adhesion of the two touching surfaces. Despite the presence of electrostatic forces arising from charged double layers in certain metals and semiconductors, this process does not play a significant role in the adhesion of nonmetallic systems (Ebnesajjad and Landrock 2008; Wake 1988).
Tissue Adhesives Based on Natural Derivatives
Fibrin Glue
Numerous studies on the use and effectiveness of fibrin sealants in many medical fields, including plastic surgery and skin grafts (Currie, Sharpe and Martin 2001), ENT (ears, nose and throat) and head and neck surgery (Staindl 1979) are also available. trauma surgery (Ochsner 1998), urology (Albala 2003; Wheat and Wolf 2009) and ophthalmology (Gauthier and Lagoutte 1989). Another disadvantage of fibrin adhesives is that they have low tissue adhesion compared to other adhesives such as cyanoacrylates and gelatin-resorcinol-formaldehyde/glutaraldehyde (GRF/). Another disadvantage of fibrin glues is the long preparation time of about 20 minutes (Conrad and Yoskovitch 2003), the need for auxiliary equipment and their ineffectiveness in high-pressure bleeding (Brennan 1991).
20 Various materials have been added to improve the properties of fibrin glue to overcome its disadvantages. Platelet factor 4 Facilitates the development of fibrin fibers by enhancing the lateral association of fibrin protofibrils.
Protein-based Adhesives
Production Methodology of Commercial Fibrin Glue Products
23 steam heating (Tissucol® /Tisseel®), and pasteurization (Beriplast®) are some of the common techniques used to inactivate viruses in commercial fibrin sealant human plasma components.
Recent Developments in Autologous Fibrin Glue Preparation
Results of the earlier research work
Clotting time is higher for each ratio of two platelet-poor plasma and lower for platelet-rich plasma, as shown in Figures 2.4 and 2.5 (Baugh et al. 2010). Previous work by the author has determined that 10% calcium chloride is the optimal concentration (Karmaker et al. 2020). The variation in adhesion properties with clotting time for different types of fibrin sealants is shown in Tables 2.3, 2.4 and 2.5, based on previous research work (Kjaergard et al. 2000).
26 Furthermore, comparative work on adhesive and bioadhesive applications on animal skin is also available (Murphy et al. 2010). Images of wounds created on the dorsum of the rat and closed by iCMBA adhesive and suture on (A) 7th and (B) 28th day after surgery.
Other Fibrin Glue Preparation Techniques
Finally, although human thrombin is being considered for use as an activator, it is not currently available for clinical use, and there is no evidence that patients will not develop an antigenic response to human thrombin. On the other hand, recombinant human factor VIII has been shown to induce antigenic responses in hemophiliacs (De Biasi et al. 1994). As a result, it is impossible to conclude that the use of recombinant human thrombin will eliminate the antigenic problems associated with bovine thrombin until more clinical studies on the effect of human recombinant thrombin are conducted.
Another problem with thrombin is that it is autocatalytic, meaning it tends to self-destruct, making it difficult to handle and store for long periods of time. Consequently, there is still a need for a convenient and practical method of preparing a bioadhesive sealant composition that presents no risk of disease transmission and no risk of causing adverse physiological reactions.
Importance of the Development of Autologous Fibrin Glue
To test the various properties of fibrin glue, known research methodologies in this field were followed, and also new techniques were developed to test other properties. In the first half of this chapter, the steps required to prepare the various solutions and plasma separation are thoroughly discussed. In the later part, the techniques used to determine setting time, adhesion strength, water content, cytotoxicity, SEM and EDS are discussed.
Preparation of Various Solutions
Anticoagulant Solution Preparation
The volume ratio of blood to anticoagulant for a 3.8% sodium citrate solution is 9:1 (Otani, Tabata, and Ikada 1998). This value was chosen because only 20 ml of blood was collected from the individual donors.
Polymer Solution Preparation
To prepare 4 mL of 0.625% w/v solution, 1 mL of concentrated polymer solution (2.5% w/v) was taken and mixed with 3 mL of deionized and sterilized water.
Calcium Chloride Solution Preparation
To prepare 4 ml of 1.25% w/v solution, 2 ml of concentrated polymer solution. 2.5% w/v) was taken and mixed with 2 ml of deionized and sterilized water.
Platelet Rich Plasma Preparation Procedure
34 It is important to avoid freeze-thaw cycles (Mitchell et al. 2005) as repeated freeze-thaw damages plasma components. Care was taken to avoid hemolysis as samples that are hemolyzed, icteric, or lipemic may invalidate some tests.
Fibrin Glue Synthesis Procedure
Platelet Rich Plasma preparation from blood
Fibrin glue from Platelet Rich Plasma
Methodology for Property Tests of Fibrin Glue
- Fibrin Clot Formation Time
- Adhesion Test
- Water Content Test
- Heat Curing Test
- Cytotoxicity Test
- SEM and EDS
Water content is an important factor affecting adhesion and is used to relate the variation of adhesion to the plasma source. One of these Petri dishes was placed on a hot plate and the temperature of the plasma solution and the hot plate was monitored using a digital thermometer. The gelation of the fibrin glue was monitored at an interval of 10 seconds as discussed earlier in the clotting time test.
The SEM images were analyzed to determine any observable difference in the structure of the fibrin glues due to the different concentrations of calcium chloride and the effect of different polymers and their concentration. In the first half of this chapter, a qualitative analysis of fibrin glue formation is discussed with relevant images.
Fibrin glue formation using different additives
To determine the performance of the developed fibrin glue, several tests were performed and their results were analyzed and compared with commercial fibrin adhesives. In the later part of this chapter, the results of the characterization tests such as setting time, adhesion strength, water content, cytotoxicity, SEM and EDS are discussed. 43 A comparison of the fibrin glue formed with 10% calcium chloride with both fresh and frozen plasma is shown in figure 4.3.
The fresh plasma gel is quite clear, while the gel formed with frozen plasma is cloudy.
Fibrin Clot Formation Time
Adhesion Strength
47 From EDS analysis, it is observed that the number of sodium ions detected for 1.25% CMC is quite high. Due to excess sodium ions, there may be some interaction between the fibrinogen and sodium ions which may delay gelation and thus decrease the overall strength with increasing concentration. Moreover, one study also showed that in the presence of sodium chloride the gelation of fibrin glue is significantly delayed (Wang, Pins and Silver 1995).
Some recent studies on fibrin glue showed 13–39 g/cm2 adhesive strength while prepared by three different approaches (Cavichiolo, Buschle and Carvalho 2013).
Water Content
Heat Curing Time
Cytotoxic Effect
SEM and EDS analysis
- Fibrin Glue formed with no polymer additive
- Fibrin Glue with 0.3125% Sodium carboxymethyl cellulose
- Fibrin Glue with 0.6250% Sodium carboxymethyl cellulose
- Fibrin Glue with 1.25% Sodium carboxymethyl cellulose
- Fibrin Glue with 0.3125% Methylcellulose
- Fibrin Glue with 0.6250% Methylcellulose
- Fibrin Glue with 1.25% Methylcellulose
- Comparison of Fibrin Glue structure with different concentration of sodium
- Comparison of Fibrin Glue structure with different concentration of methylcellulose
In Figure 4.14, two magnification levels, one at 500x and one at 1000x, of fibrin sealant with 0.3125% sodium carboxymethylcellulose are shown, along with EDS data. In Figure 4.15, two magnification levels, one at 500x and one at 1000x, of fibrin sealant with 0.6250% sodium carboxymethylcellulose are shown, along with EDS data. In Figure 4.16, two magnification levels, one at 1000x and one at 3000x, of fibrin sealant with 1.25% sodium carboxymethylcellulose are shown, along with EDS data.
Comparison of the structure of fibrin glue with different concentrations of sodium carboxymethyl cellulose sodium carboxymethyl cellulose. In Figure 4.20, SEM images of fibrin glue with different concentrations of sodium carboxymethyl cellulose are shown at 1000x magnification.
Cost Comparison of Fibrin Glue
Although the developed fibrin sealant is promising, more research is needed to develop it even further and to eliminate several disadvantages of this research.
Conclusions
In general, fibrin glue formulated with methylcellulose had higher adhesion strength compared to that formulated with sodium carboxymethylcellulose. The water content data suggested that fibrin glue from freshly prepared plasma contains water in the range of 1 to 2%. Furthermore, since the fibrin glue will be used inside the human body, other cytotoxicity tests may need to be performed before actual clinical trials.
65 In summary, the process of making fibrin glue from human plasma is simple, however, it is very difficult to understand the inner workings of the process from direct analytical tests. It is clear from the various test data that methylcellulose is the best polymer to use as an additive in fibrin glue.
Recommendations
Use of fibrin glue in correction of Pollybeak deformity: A preliminary report. Archives of Facial Plastic Surgery. Use of fibrin glue in skin grafts and tissue-engineered skin substitutes: A review. Plastic and Reconstructive Surgery. Use of a fibrin glue (Tissucol) for treatment of perforated or pre-perforated corneal ulcer]. Journal français d'ophthalmologie.
Development of natural fiber-supported high-strength autologous fibrin glue from human plasma." Journal of Adhesion Science and Technology. Preparation of fibrin glue: the effects of calcium chloride and sodium chloride." Materials science and engineering C.
