Decortication of Eichhornia crassipes (Pontederiaceae) Fibers for the Production of Multifilament Non-Absorbable Surgical Suture

Water hyacinth (Eichhornia crassipes) is one of the worst aquatic weeds in the Philippines [1]. Its rapid infestation is due to its ability to double its number in as fast as 12 days. It has a detrimental impact on agriculture, economy, environment, recreational activities, water quality and quantity, and human health.

Despite it being one of the world’s most destructive weed, it exhibits antibacterial, antifungal and anti-algal properties [2]. Crude extracts of the plant showed growth inhibition of Gram-positive bacteria, Gramnegative bacteria, yeast, green microalgae, and cyanobacteria. Moreover, the Philippine Research Textile Institute (PTRI) is developing its use in domestic textile to trim the industry’s import of synthetic-based fibers [3]. The plant’s fibers are used as raw materials for the production of handicrafts, furniture’s, and tableware’s.

Due to its relatively strong and flexible fibers, this study aims to produce a natural multifilament non-absorbable surgical thread out of water hyacinth.

Methodology

Materials and equipment: Eichhornia crassipes with 20-inch petioles were collected. After formal plant identification, the leaf blades and roots of the plants were removed, leaving the petioles behind for the decorticating process via a decorticating machine. After decortication, the fibers were then air-dried and manually braided to produce the suture. Samples were categorized according to quantitative composition: 15 strands of fiber and 30 strands of fiber; according to relative size: Size 1 Silk Suture, Size 4-0 Silk Suture.

The materials: 15-strand and 30-strand braided water hyacinth threads, silk sizes 1 and 4-0, needle holder, surgical needle, thumb forceps, mayo scissors and iris scissors, were packed in aluminum foil and sterilized using an autoclave (121°C at 15 psi). On the other hand, wooden clamps, metal hooks, and the weight receptacle composed the improvised tension meter.

Six adult male albino rabbits (Oryctolagus cuniculus), each weighing approximately 2 kg, were utilized in the experiment. The animals were kept in a room at 23-27°C with 75% relative humidity. Ventilation rate was maintained at 10-20 air changes/hr by mechanical means. The fluorescent lighting provided followed a diurnal cycle. An acclimatization period of at least 5 days was allotted.

With respect to the animal welfare policies, evaluation of the scientific use, handling and care of the adult male albino rabbits was conducted by the Institutional Animal Care and Use Committee of the University of Santo Tomas. All experimental methods, reagents, and equipment to be used on the rabbits were assessed by the committee before proceeding with in-vivo scientific tests.

Physical properties of the water hyacinth threads

Diameter: The number of fibers of each braided suture was estimated to match the diameter of the silk sutures. A microscope with an ocular micrometer was used to measure the diameter of the braided plant fibers.

Tensile strength test: Six inches of both the water hyacinth sutures and silk sutures were tested using the improvised tension meter. Three trials were done for each sample to determine the mean tensile strength.

Knot tensile strength test: Six inches of both the water hyacinth sutures and silk sutures were tested using the improvised tension meter. Samples contained four throws of square knot in the middle to determine knot tensile strength. Re-tests were done when suture breakage did not occur beside the square knot. Three trials were done for each sample to determine mean knot tensile strength.

Heavy metal content determination: Atomic absorption spectrophotometry was used to determine the presence of heavy metals. The test portions were dried and then incinerated and turned to ash at 450°C under a gradual increase (<50°C/h) in temperature. Then, 6 M hydrochloric acid (HCl) was added and the solution was evaporated to dryness. It was digested completely until dissolved using 0.1 M nitric acid (HNO₃) and filtered out. Distilled water was added. After which, atomic absorption spectrophotometer was used to analyze the analyte (AOAC Official Methods of Analysis).

Pharmacological properties of the water hyacinth threads

Sterility test (Direct transfer method): The autoclaved water hyacinth suture samples were tested for their sterility by direct transfer method (USP 30/NF 25 2007). Threads measured 30 cm each. Eight sutures of the 15-strand and eight sutures of the 30-strand were required to complete one run of the test.

Test for aerobic and anaerobic bacteria: The sample sutures were transferred directly into the fluid thioglycollate broth. Other tubes of fluid thioglycollate broth inoculated separately with Staphylococcus aureus and Pseudomonas aeruginosa served as the positive controls. A tube of non inoculated fluid thioglycollate broth served as the negative control. All tubes were incubated at a temperature of 37°C then observed for turbidity for 14 days (USP 30/NF 25 2007).

Test for fungi: The sample sutures were transferred directly into the Trypticase soy broth. Another tube of Trypticase soy broth inoculated with Candida albicans served as the positive control. A tube of non-inoculated Trypticase soy broth served as the negative control. All tubes were incubated at 37°C and observed for turbidity for 14 days (USP 30/NF 25 2007).

Growth promotion test for aerobes

The sample sutures were directly transferred into four tubes of fluid thioglycollate broth. Two tubes were inoculated with S. aureus (P. aeruginosa. Another two tubes of fluid thioglycollate broth inoculated separately with S. aureus and P. aeruginosa served as the positive controls. A tube of non-inoculated fluid thioglycollate broth served as the negative control. The tubes were incubated for not more than 3 days (USP 30/ NF 25 2007).

Growth promotion test for fungi

The sample sutures were directly transferred into two test tubes of Trypticase soy broth. These were inoculated with C. albicans (C. albicans and served as the positive control. A tube of non-inoculated Trypticase soy broth served as the negative control. The tubes were incubated for not more than 5 days.

Surgery and skin irritation test

Prior to surgery, the rabbits were divided into two groups. Skin integrity was checked. The fur was shaved from the dorsal area of the trunk to expose the skin. Caution was observed to avoid abrasion. Before incision, 15 mg/kg ketamine 2 mg/kg midazolam was administered intramuscularly to the test animals. After onset of the drug through physical inspection, the skin on the dorsal area of each rabbit was incised twice, each having a 6 cm incision parallel to each other. Group A of the test animals was sutured with 15 strand water hyacinth suture and silk 4-0. On the other hand, Group B was sutured with 30 strand water hyacinth suture and silk 1. A triangular-tipped surgical needle was used to employ a simple interrupted pattern of stitching having a 4 mm suturing interval. Incision wound was treated with povidoneiodine solution and secured with sterile gauze. Daily inspection with gauze replacement and wound cleaning was done for two weeks.

Inspection included grading of erythema and edema, detection of pus discharge and wound dehiscence [4]. After 7 days, euthanasia by cranial concussion was done to harvest the pelt and subject it to wound tensile strength measurement and histopathology. (Tables 1 and 2)

Table 1: Criteria for Erythema and Eschar Formation (OECD Guidelines 404, [4]).

Table 2: Criteria for Edema Formation (OECD Guidelines 404, [4]).

Euthanasia

Cranial concussion (Stunning): The rabbit was suspended by its hind legs, grasping around both hocks with the left hand. A cudgel was used to deliver a single, heavy, sharp blow to the back of the skull. Cessation of respiration, heartbeat, and reflexes were noted to indicate irreversible death.

Removal of the suture: The suture was cut under the knot, close to the skin surface. When pulled from the wound, the previously exposed and contaminated portion of the suture does not travel back through the wound.

Wound tensile strength test: Twelve square centimeters of excised skin length containing the sutured incision was subjected to wound tensile strength test. Tensile strength was determined when the first sign of wound opening was seen. Weight of sand used in the procedure was measured using weighing balances with 1 g and 5 g. calibration marks.

Histopathology

Preparation of specimens for tissue processing: Out of the 6-cm incision made on each rabbit, 2 cm were taken for tissue processing. After cutting the pelt into its appropriate size, it was further divided into 2-mm strips then immersed into 10% neutral buffered formalin solution. The specimens were then submitted to a diagnostics laboratory for tissue processing.

Histopathologic analysis: After tissue processing, the specimens which were mounted onto microscope slides were then submitted to a histopathologist for examination of antigenicity, extent of wound healing and the formation of granulation tissue were included.

Physical properties of the water hyacinth threads diameter

Diameter of the 15-strand water hyacinth suture and silk size 4-0 are considered statistically different (t-value=0.000). Also, diameter of 30-strand water hyacinth suture and silk size 1 are considered statistically different (t-value=0.000). Diameter means, standard deviations, and t-values are shown in Table 3.

Table 3: Diameter Measurement Means, Standard Deviations, and t-value.

Tensile strength test

Tensile strength of the 15-strand water hyacinth suture and silk size 4-0 are considered statistically different (t-value=0.002). Also, tensile strength of 30 strand water hyacinth suture and silk size 1 are considered statistically different (t-value=0.004). Tensile strength means, standard deviations, and t-values are shown in Table 4.

Table 4: Tensile Strength Means, Standard Deviations, and t-value.

Knot tensile strength

Knot tensile strength of the 15-strand water hyacinth suture and silk size 4-0 are considered not statistically different (t-value=0.324). Also, knot tensile strength of 30-strand water hyacinth suture and silk size 1 are considered not statistically different (t-value=0.065). The knot tensile strength means, standard deviations, and t-values are shown in Table 5.

Table 5: Knot Tensile Strength Means, Standard Deviations, and t-value.

Pharmacologic properties of water hyacinth fibers sterility test

As shown in Table 6, both samples are positive on the growth promotion for aerobes and fungi. On the other hand, one 30-strand water hyacinth suture is positive on the test for the presence of fungi.

Table 6: Sterility Test Results of Autoclaved Water Hyacinth Sutures.

Wound tensile strength

Wound tensile strength of the 15-strand water hyacinth sutured wound and silk size 4-0 sutured wound are considered not statistically different (t-value=0.095). Also, wound tensile strength of 30 -strand water hyacinth sutured wound and silk size 1 sutured wound are considered not statistically different (t-value=0.647). The means, standard deviations, and t-values are displayed in Table 7.

Table 7: Wound Tensile Strength Means, Standard Deviations, and t-value.

Skin irritation and drag test: Skin irritation was not observed in all rabbits after surgery. During surgery, it was noted that the 15-strand water hyacinth thread had moderate drag compared to the silk sutures having no drag. The 30-strand water hyacinth thread had the most drag but yielded a better wound closure compared to the silk control. Strand breakage for all threads was not observed along the course of surgery procedure (Table 8).

Table 8: Scoring of Erythema and Edema for Six Male Albino Rabbits.

Presence of wicking and wound dehiscence

Wound exudates from both threads were absent. Hence, wicking did not occur. Wound dehiscence was noted on two incisions sutured with Silk 4-0 and one incision with 15-strand (Table 9).

Table 9: Rabbits that exhibited with wound dehiscence.

Histopathology

Examination revealed that wounds sutured with 15-strand water hyacinth underwent moderate to severe inflammation while wounds sutured with silk 4-0 had shown mild to moderate inflammation. Examination on wounds sutured with 30-strand water hyacinth and silk 1 were inconclusive.

One wound sutured with 15-strand water hyacinth and all three wounds sutured with silk 4-0 exhibited neovascularization. However, neovascularization was present on one wound sutured with 30-strand and another wound sutured with silk 1.

Excessive formation of fibrous connective tissue were observed on one wound sutured with 15-strand water hyacinth, three wounds with silk 4-0, and one wound with silk 1.

Diameter and tensile strength data showed that the differences of the means of water hyacinth sutures and silk sutures were statistically significant (t<0.05). However, knot tensile strength and wound tensile strength data showed that the differences of the means of water hyacinth sutures and silk sutures were not statistically significant (t>0.05). Also, cadmium, silver, mercury, lead, and copper were considered undetected in plant fiber. This passes the water hyacinth suture in the physical aspect of the study. Edema, erythema, and wicking were absent on both silk and water hyacinth sutures. Wound dehiscence was seen in 3 sutured wounds; 2 of which were with silk suture and the latter with the water hyacinth fiber suture. Conclusively, the decorticated fibers from E. crassipes can be utilized to produce multifilament non-absorbable sutures with physical properties comparable to silk.