Mechanical and in-vitro studies of biodegradable chitosan/cissus quadrangularis coated flax sutures for anterior cruciate ligaments repair

Abstract

In this investigation, the mechanical and In Vitro studies of chitosan/cissus quadrangularis (CHCQ) coated braided flax sutures are carried out to explore their application towards repairing the anterior cruciate ligaments (ACL). The 5% (w/w of suture) mixture of Chitosan (CH) and quadrangularis extract (CQ) was applied on the braided suture. Chitosan (CH) and quadrangularis (CQ) mixtures were prepared in different weight ratios such as 4:1 (CHCQ41), 3:2 (CHCQ32), 1:1 (CHCQ11), and 2:3 (CHCQ23) for experimental work. These mixtures were coated on the suture using the pad-batch technique. GC-MS analysis found that cissus quadrangularis extract containing Caffeine and Phytol with the ratio of 3.20% and 15.98%. These petrochemicals are essential for muscle relaxation, pain reliever, and strengthening the immune system. Through Scanning electron microscopy analysis, It was found that with the incorporation of chitosan polymer, the surface fibrils in the suture are wrapped into the core and shown as an effective biocomposite suture. Most of the samples have displayed ultimate tensile stress between 2846 N to 2865 N, which is higher than the human ACL. The Stiffness of the suture samples varied from 437 to 471 N/mm, and the elongation of the suture samples was between the range of 34%–36%. Similarly, the knot pull strength of the suture samples varied from 241 to 243 Mpa. CHCQ11&CHCQ23 has shown maximum functional performance among the different suture samples due to higher Cissus quadrangularis proportion. MTT on cell viability assay sample CHCQ11&CHCQ23 have shown higher cell growth and proliferation by identifying its optical density value after the fifth day was 0.63 and 0.65. Similarly, the hemolytic percent is also higher for CHCQ11&CHCQ23 with 0.7 and 0.8 Hg%.

Introduction

Sportspersons very often suffer due to ligament rupture. Anterior Cruciate Ligament (ACL) is a kind of knee injury most commonly happens to athletes. Ligaments are formed by bundles of connected dense fibres that support connecting bone and joints. Ligaments are naturally created and contain 90% of type I collagen and 10% type III collagen. ¹ Ligaments must have high tensile strength and elasticity for maintaining the joint’s stability, which often undergoes physiological movement. Sometimes due to intense physiological activities of the sportsperson lead to ligament injury. As reported by Docheva et al., ² it is essential to replace the ligaments when severely damaged. The ACL rupture affects the individual’s mobility and causes pain and discomfort. ³ According to Smith et al., ⁴ ACL injury rates are 2.8–3.2 per 10,000 athletes. No reliable surgical procedures are available to heal the ACL, and most of the time, ACL injury leads to long-term pain, tendonitis, and muscle atrophy. ⁵ ACL reinforcement is an alternative way for sustaining an ACL graft in a synergistic load-sharing manner with primary stress on the graft and a high strength special suture as protective safety belt. ⁶ In addition, if not surgically restored, incomplete ACL injuries in children may proceed to a complete tear, with poor functional results with subsequent meniscal and chondral damage. ⁷ Hamstring grafts (HI) is a popular reconstructive surgery executed to ACL fixation through high-strength Sutures. ⁸ In the case of HI, with the support of specially made sutures, the lateral cortex of the distal femur is secured endoscopically or by other means. This treatment method ensures the immediate mobility of ACL injured patients.^9,10 The Ligament Advanced Reinforcement System (LARS), made out of non-biodegradable polyethylene terephthalate (PET) filaments, has been used commercially for ACL injury. ¹¹ Though these LARS ligaments are biomimetic, it lacks certain properties such as osteoconduction and osteoinduction. In addition to this, It affects the growth of the bone on the implants. At present, the ACLs have been managed by surgeries using semi-synthetic and non-absorbable sutures due to their higher strength. Clinical Surgery treatment took months to cure without any side effects caused by the synthetic and semi-synthetic Sutures. Researchers are working to develop biocompatible sutures for various surgical treatments using fibres such as silk, wool, collagen etc. ¹² Therefore, an ideal suture should be biodegradable, biocompatible, and possess high tensile strength. ¹³ Moreover, treatment like HI surgical sutures require very high strength structured special sutures to hold the surgical organ intact, which could not be produced through general suture production technique. The use of suture tape augmentation in the repair/reconstruction of medial knee injuries, ulnar collateral ligament repair/reconstruction, and ACL repair/reconstruction with allograft. ¹⁴ María et al. Studied the close looped graft suturing towards ACL reconstruction using mature porcine tibiae and flexor digitorum profundus tendons that were harvested from freshly slaughtered 6-month-old pigs and found its ultimate tensile strength was between 946.5 N to 1020.1 N. ¹⁵ Similarly, Harvey studied the mechanical properties of graft in the reconstruction of the anterior cruciate ligament and found that the ultimate load and stiffness was found 2160 N and 242 N/mm. ¹⁶ Braiding is one of the 3D structure formation techniques with the inter-plaiting of three or more orthogonal sets of yarn to form high strength and stiffer structures like cables, hoses, industrial belts, and surgical sutures. ¹⁷ Over the last few decades, the use of natural products for pharmacological purposes has increased over the world. ¹⁸ Flax fibers, as an example of biologic material, have been considered due to their intrinsic mechanical properties and biocompatible with the human tissues. ¹⁹ Flax fibres have been considered the important biomaterials, including sutures. These include low density, comparable tensile properties, high abrasion resistance, non-irritation to skin, less health risk, and renewability, recyclability, and biodegradability. ²⁰ Flax fibres have thus been considered for various biomedical applications including biocomposites for tissue engineering, sutures, and biological implants because they are biocompatible with human tissues to a great extent^21,22. Baley et al. studied the mechanical properties of Flax and jute fibres, found that both fibres are relatively similar in mechanical properties and effectively used in biocomposite fabrication. ²³ Alkali pretreatment significantly improves the mechanical properties of the natural fibre. Amutha and Kumar have done a comparative analysis of raw and alkali-treated Acacia Concinna fibre and found an improved crystallinity index than the raw fibre between the range of 27.5%–35.6%. ²⁴ Similarly, Senthamaraikannan and Kathiresan studied the physical, chemical, tensile, crystalline, thermal, and surface morphological properties of raw and alkali-treated Coccinia Grandis. L Fibers and found that the alkali treatment has improved the tensile strength, crystallinity index, thermal stability, and roughness of the fibre. ²⁵ Plants are an important source of novel therapeutics. Though many plants possess bioactive molecules, only a small percentage have been explored^26,27. Cissus quadrangular uses a folk-fore medicine in the Indian subcontinent and has many proven medicinal properties. ²⁸ Phytochemical studies revealed that Cissus quadrangular found several compounds such as flavonoids, triterpenoids. Similarly, Chitosan is a non-toxic and biodegradable biopolymer, and it is obtained from exoskeletons of crustaceans, arthropods, mollusks, and some fungi’s cell walls. Sang et al. ²⁹ found the chitosan pretreatment on cotton fabric improved the antimicrobial activities against Staphylococcus aureus. Chitosan has been utilized in the field of textile for its biocompatibility, biodegradability, nontoxicity and antimicrobial activity^30–32 and also used in drug delivery formulations^33,34. Shanmugasundaran et al. Studied the Tetracyline hrdrochloride type drug release bahaviour of chitosan-coated cotton yarn and found that the amount of drug absorbed and released is more by chitosan coated cotton yarn than untreated samples, which is due to polycationic nature of chitosan. ³⁵ Giridev et al. ³⁶ treated the wool fabric with Chitosan and found the higher antimicrobial activity of treated fabric for a longer period. The Flax fibre possesses higher tensile strength and modulus comparable with silk-based sutures. ³⁷ Abdel et al. ³⁸ Studied cell growth and wound healing property of the chitin-glucan wound dressing using animal model and found that this composition have not shown any cytotoxicity against mouse fibroblast cells and exhibited excellent surgical wound healing ability. Similarly, the cell viability and proliferation studies were conducted for hemp fibre-based 3D sacrificial porogen template, and Cytotoxicity performed assay indicated that analyzed extracts exhibited higher cell densities and proliferation capacity at 3-days incubation point. ³⁹ Braiding is one of the textile structures which is more suitable for producing suture configuration. The braided sutures possess high abrasion strength, and the suture anchor characteristics are improved. ⁴⁰ Multi-functionalization studies were conducted to achieve more than one functional properties of textile material. Javed et al. ⁴¹ Studied the multi functionalization of linen using Chitosan and Tamarindus and found that wrinkle recovery, antibacterial activity, ultraviolet protection, and antioxidant properties were found. Similarly, combination of Chiotsan with Cissus quadrangular provides the antimicrobial activity along with wound healing properties to the suture material. There has been no previous research on the influence of independent braided suture tape reinforcement with wound medicine on the biomechanical stability of HS tendon graft constructions for ACL restoration. This research aimed to develop the bio-degradable braided suture tape using flax fibre coated with the mixture of chitosan/Cissus quadrangular extract for ACL rupture. Most of the selected parameters falls under natural source for the fabrication of braided suture tape to obtain the combined effect of tensile strength, antimicrobial property, biodegradability, non-toxicity, and wound healing. The chitosan polymer not only provides the antimicrobial activity to the Suture but also can act as an integrator of flax fibre with Cissus quadrangular ⁴² .

Materials

Raw flax fibre and necessary chemicals were purchased from Samy and co. Ltd Coimbatore. Individual Flax fibres were cut into an average length of 9 cm and a mean diameter of 0.02 mm. The tenacity of the bundle fibre falls between 6.5 to 8 g/denier, and elongation at break was 1.8% (dry) and 2.2% (Wet). The moisture regain of the fibre is around 12%. The fibres were braided using 12 spindle tabletop braiding machines at the yarn manufacturing facility of the Gandhigram Rural Institute, India. The output suture diameter was made around 4 mm. The image of the braiding machine and braided flax Suture is shown in Figure 1. The Cissus Quadrangularis (CQ) plants were collected from the campus of Gandhigram Rural Institute. The Chitosan with molecular weight (Mn) of 15 kDa and degree of deacetylation (DDA) 95.3%, was obtained from Central Institute of Fisheries Technology, Kochi,India.

Figure 1.

Braiding process. (a) Braiding machine, (b) braided Flax suture.

Extraction of herbal plants

The Cissus Quadrangularis (CQ) plant (Figure 2) was collected from a single source throughout the experiments in order to avoid batch-to-batch variation. The stem of CQ was washed thoroughly with double distilled water; shade-dried. The stem of the CQ plant was cut, well dried, and powdered. 100 G of powdered CQ was mixed with 400 mL of methanol using the soxhlet apparatus and filtered through Whatman filter paper (No 41). ⁴³ 4g of sodium silicate was used as a sedimentation agent and subsequently dried to get the powder form of CQ crude mixture.

Figure 2.

Cissus quadrangularis plant.

Pretreatment by alkaline method

120 cm long braided flax suture weighed as 0.028 mg using a digital balance. Material to liquor ratio was maintained at about 1:20 ratio (w/v). 0.028 g of the non-ionic wetting agent (Egyptol) was added to decrease the surface and interfacial tension of the braided suture. 0.168 g of sodium hydroxide and 0.112 g of sodium carbonate were added to increase the alkalinity. It was then allowed to stand for 3 h with intermittent stirring using the glass rod

Preparation of chitosan/cq composite coating

The mixture of Chitosan (CH) and CQ extract of 5% (w/w of Suture) was applied to the sutures having CH and CQ ratios such as 4:1(CHCQ41),3:2(CHCQ32),1:1(CHCQ11), and 2:3(CHCQ23) for experimentation. The Chitosan mixture was prepared by dissolving 5g of Chitosan (Commercial grade water-soluble chitosan, deacetylation = 95.3%) within 100 mL of 0.35 m acetic acid; subsequently, 0.5 m of NaOH was added for neutralizing the solution. ⁴⁴ Parallelly, 5g of CQ extract was prepared in 100 mL of boiling water, and both the mixtures are taken as per indicated ratios of sample CHCQ41,CHCQ32,CHCQ11, and CHCQ23, but the total add-on was maintained as 5% of the weight of the sample. ²⁸ These different combinations of CH/CQ mixtures were coated on flax braided structures using the pad-batch technique. Subsequently, the coated structures were treated with 80% (v/v) ethanol solution for 2 h to enhance the chemical fix on the fibre surface. Table 1 depicts the composite coating specifications of the braided suture samples.

Table 1.

Braided Flax suture samples coating specification.

S.No	Sample code	% of chitosan on 5% (w/w) of the suture	% of Cissus Quadrangularis on 5% (w/w) of the suture
1	CHCQ41	80	20
2	CHCQ32	60	40
3	CHCQ11	50	50
4	CHCQ23	40	60

Characterisation

Scanning electron microscopy

All the braided suture samples were sputter-coated with gold to analyze the surface characteristic of the samples^32,45. FlexSEM 1000 instrument with a working voltage of 10 kV was utilized for testing. This analysis was done to understand the coating effectiveness of the fibre sample

Fourier transform infrared spectroscopy

FTIR spectroscopy was executed for the analysis of the chemical structure present in the fibre sample. During testing, the measurement was taken between the ranges of 500 to 4000 cm⁻¹

Gas chromatography-mass spectrometry analysis

The GC-MS analysis was carried out for the crude extract of CQ using the GC Clarus 500 Perkin Elmer system. This instrument was equipped with column elite, 70 eV operating electron impact modes. Helium gas was used as carrier gas with a constant flow of 1 mL/min. The inlet temperature was set at 250°C; Oven temperature was 110°C with the increase mode of 10°C/min, endpoint temperature was up to 280°C. The diluted plant extract sample of 1 mL was injected, and mass spectra were taken with the scan interval of 0.5s.

Tensile studies of the suture

The ultimate Tensile stress(N), Stiffness (N/mm), Yield strain (%),Elongation (%) and simple pull knot strength (Mpa) were measured using universal testing machine (INSTRON model 1405) at an extension rate of 5 mm/min. The normal yarn testing jaws were used for this study. Due to the surface treatment, suture slippage may happen during the tensile testing. The test material was gripped with the support of a metal pin to overcome the slippage during testing. The tensile testing conditions were as follows gauge length is 10 inches (25.4 cm), crosshead speed was 5 mm/min

Antifungal activity

Antifungal activity of coated sutures was determined as agar-well diffusion method. ⁴⁶ The following fungal strains, such as Microsporum fulvum, Trichophyton mentagrophytes, Trichophyton rubrum, and candida albicans were used for the antifungal study. ⁴⁷ Sabouraud dextrose agar (SDR) medium was used to maintain the fungal strain at room temperature to prepare inoculums. The sterile loop was utilized to transfer the fungal strain spores into these inoculums, and its concentration was kept as 105 CFU/ml. The wells of 6 mm diameter of PDA plate were filled with the test specimen. Control experiments were carried out under similar conditions using commercially available fungicides, Nystatin and Griseofulvin were used as a positive control and DMSO as a negative control. All the Petriplates including treatments and controls were allowed to diffuse at room temperature for 2 h and then incubated at room temperature (28 ± 2°C) for 72 h. After incubation, the antifungal activity of extracts was expressed in terms of diameter of zone of inhibition.

Cell viability and proliferation studies

MTT assay was carried out with Human osteoblast-like cells (MG-63) to understand the cell viability of the braided Flax suture. Human osteoblast-like cells (MG-63) were purchased from a cell bank, National Centre for Cell Science (NCCS), Pune, India. The Flax braided sutures were sterilized with 70% ethanol and UV light for 30 min. Then MG-63 cells were treated with suture samples for 48 h. The MTT assay is a colorimetric assay for assessing cell metabolic activity based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan crystals are dissolved using a solvent, and the resulting colored solution is quantified by measuring absorbance at 500–600 nm using a spectrophotometer. The darker the solution is, the indication of the higher number of viable, metabolically active cells. ⁴⁸ There were five well containing the braided Flax samples in different surface treatment ratios and one control sample. Each well was seeded with 1000 μL of medium containing approximately 1x10 ⁴ MG 63 cells. The plates were incubated at 37°C with 5% CO₂, and 5 mg of MTT solution was added with the well. After the regular interval of first,third^, and fifth days, the sutures were dried well, and the insoluble purple coloured formazan crystals were solubilized using dimethyl sulphoxide. The viability of the cells in culture depends on the reduction level, which is measured as the optical density of coloured formazan crystals solution at a wavelength of 570 nm. The absorbance was proportional to the number of cells on the composite. Three replicates of each sample were prepared at each time point. The cell viability was calculated using the following formula

$$Cellviability (\%)=\frac{\left(Optical density of treated sample-Optical density of medium\right)}{ \left(Optical density of control sample-Optical density of medium\right)}\times 100$$

Blood compatibility

Blood compatibility is the most crucial property of biomedical materials. The human blood sample was collected from Bose clinical laboratory. The blood was loaded in a sterile test tube containing 3.8% sodium citrate. Then the blood was diluted with phosphate buffer saline (PBS) in the ratio of 1:20. All the coated Flax sutures were treated with UV light for 30 min and stabilized with PBS for 24 h at 37°C. The samples were immersed in 2 mL of blood diluted with PBS solution. These samples were incubated at 37°C for 60 min and centrifuged at 3000 r/min for 10 min, and the supernatant was collected. The optical density value (OD) was measured through a spectrophotometer at 545 nm. The blood diluted with PBS (no hemolysis) was taken as a negative control, and the blood with Triton X (100% hemolysis) was taken as a positive control. The hemolytic percentage was estimated using the following equation

$$Hemolysis (\%)=\frac{\text{A}540\text{ of RBC treated sample}-\text{A}540\text{ of buffer}}{\text{A}540\text{ of triton X}100\left(1\text{\%}\right)-\text{A}540\text{ of buffer}}\times 100$$

Results and discussion

Gas chromatography-mass spectrometry analysis of cissus quadrangularis aqueous extract

GC-MS study showed the presence of the different chemical constituents in CQ extract. Table 2 demonstrates the essential derivatives are present in it, such as phenolic compounds, fatty alcohols, and carboxylic compounds that possess antioxidants, anti-inflammatory compounds, and hypoglycemic activities^49,50. In this report, Caffeine is one of the vital compounds, which present in many plant extracts. It is one of the lipophilic compounds that can decrease fatigue, muscle relaxation, and tension reliever. ⁵¹ Similarly, another important reported compound is phytol, which is the key source for vitamin E production and can strengthen the immune system.

Table 2.

GC-MS analysis of Cissus quadrangularis (CQ).

S.No	Retention time (min)	Mass(g)	Compound name	Compound (%)
1	4.05	160	Pentane,1,1-diethoxy- (CAS 3658-79-5)	1.85
2	9.926	150	Ethanone, 1- (2-hydroxy-5-methylphenyl)	0.25
3	23.32	194	Caffeine	3.20
4	21.78	274	Phenol, 2,4-bis(1,1-dimethylethyl)	23.17
5	25.62	278	N-Methyl-1-adamantaneacetamide	34.50
6	25.86	256	n-Hexadecanoic acid	25.63
7	26.4	284	Hexadecanoic acid, ethyl ester	7.57
8	28.79	296	Phytol	15.98
9	26.7	—	Unknown	15.30

Scanning electron microscopy

The surface morphology of the raw Flax and CH/CQ coated Flax suture was examined in a longitudinal direction using a scanning electron microscope at different magnification; it is shown in Figure 3 and4. In Figure 4, the evenly distributed surface particles are visible, and it is evident that the treatment was effective. Due to the incorporation of chitosan polymer, the surface fibrils are wrapped into the core of the suture and shown as an effective biocomposite.

Figure 3.

Scanning electron micrographs of Raw braided Suture –a (82X), b (580X).

Figure 4.

Scanning electron micrographs of CH/CQ coated braided Suture of sample CHCQ23–a (106X), b (536X).

Fourier transform infrared spectroscopy analysis

The FTIR spectra recorded for the raw and coated Flax sample CHCQ23 with ATR mode and its spectra diagrams are shown in Figure 5. The FTIR spectrum of Chitosan/CQ coated Suture has shown the peak in the following positions such as 3236 cm⁻¹,1728 cm⁻¹,1602 cm⁻¹,1041 cm^−1, and it has a similarity of the research work done by Devipriya et al. ⁵² 3236 cm⁻¹ indicated the carboxylic acid O-H stretching due to the presence of cellulose in CQ. A small peak at 1602 cm⁻¹ is attributed to the presence of carboxyl stretching of C-O, which is an acetyl group of hemicellulose in CQ. ⁵³ The peak 1602 cm⁻¹ confirmed the NH stretching and the C-O and CH₂ stretching of chitosan. ⁵³ The band 1041 cm^-1 corresponds to alkoxy C-O bond bending vibration. ⁵⁴ Similarly, the presence of Chitosan is also revealed by its typical peak shown only by treated samples at 1580 cm−1 which is assigned to N–H bending.^44,55

Figure 5.

FTIR analysis of Raw and treated Flax Suture of CHCQ23.

Mechanical property

The mechanical properties of the suture material are essential because of their use in internal and external surgeries. From Table 3, it is clear that the Flax suture prepared using CH/CQ embedded with plant extract has better mechanical properties than the Human ACL. The final diameter of the suture samples was measured as 4 mm. The comparative mechanical properties of the braided sutures are shown in Table 3. Flax suture possesses ultimate tensile stress between the range of 2846 N–2865 N, and the stiffness values are 437 N/mm to 471 N/mm, yield strain (%) percentage between the range of 12.38–12.98, elongation (%) between the range of 34.34%–35.72%, and Simple knot pull strength (MPa) between the samples in the range of 241.32 Mpa 242.72 Mpa. These values are higher than the Human ACL calculated by Woo et al. ⁵⁶ In addition to this, due to the braided configuration, the structural integrity of the sutures was found good. All the test samples reflected the same kind of mechanical behavior since the treatment has not modified the morphological components of the fibre.

Table 3.

Mechanical properties of 4 mm diameter braided Flax sutures and human ACL taken from Woo et al. ⁵⁶

Parameters	Human ACL	Control sample	CHCQ41	CHCQ32	CHCQ11	CHCQ23
Ultimate tensile stress(N)	2160 ± 153	2846 ± 73	2854 ± 85	2858 ± 94	2862 ± 68	2865 ± 77
Stiffness (N/mm)	243 ± 28	437 ± 18	464 ± 13	453 ± 14	469 ± 9	471 ± 15
Yield strain (%)	10 ± 1.4	12.43 ± 0.9	12.54 ± 1.04	12.38 ± 1.05	12.45 ± 1.1	12.91 ± 1.04
Elongation (%)	33	34.34	35.43	34.54	35.67	35.72
Simple knot pull strength (MPa)	—	242.34	243.42	241.32	243.23	242.75

Biological characteristic of Suture

Antifungal characterization

The agar well diffusion assay showed the inhibition zone against the different microorganisms. Figure 6, depicted the zone of inhibition of all the suture samples. Samples CHCQ11&CHCQ23 have shown higher inhibition than other samples. This is presumed that the CHCQ11&CHCQ23 samples contain a greater amount of Cissus quadrangularis content than chitosan content, which leads to this drastic improvement in antifungal activity. The Cissus quadrangularis extract possessed higher antimicrobial and antifungal resistance due to the presence of phytochemical groups.

Figure 6.

Antifungal activities against different microorganism.

MTT test

MTT on cell viability assay was performed to find out the effect of chitosan/CQ treatment cell viability. The cell reference used in this study was MG-63. Figures 7 and 8 show the MTT results. After the first,third, and fifth day of culture, a gradual increase in cell growth and proliferation was found on the Flax braided sutures. There would be a gradual increase in cell proliferation from first day to fifth day of the incubation period for all the samples. Among the samples, higher cell growth and proliferation were found for samples CHCQ11&CHCQ23. It has been inferred that samples CHCQ11&CHCQ23 have a higher proportion of Cissus quadrangularis content than other samples. It is further understood CHCQ11&CHCQ23 samples contain a low percentage of Chitosan, which minimizes the moisture absorbency comparative to other samples. This relatively low moisture absorbency supported the higher zone of inhibition. The statistical difference between the treated samples towards cell proliferation was significant (p < .001). Figure 8, Shows the Optical imaging of the cell morphology and cell proliferation. It is evident that the cell growth is higher for CHCQ11&CHCQ23 samples after fifth day.

Figure 7.

MTT assay of MG-63 cells in direct contact with the examined systems after 1, 3 and 5 days in terms of optical density values.

Figure 8.

Optical imaging of the cell morphology after fifth day in different treated samples (a)- control sample (b)-CHCQ41, (c)- CHCQ32 (d)- CHCQ11 (e) CHCQ23.

Hemolytic activity

The hemolytic activity is an essential study to understand the compatibility of medical devices when in contact with blood. ⁵⁷ The interaction of implantable materials, like sutures with blood, can result in the release of hemoglobin, which is influenced by the surface of the material. ⁵⁸ The Figure 8 shows the hemolytic percent of all the test samples with reference to the rubber and medical steel. The hemolytic activity of all the suture samples was almost equal, but the samples CHCQ11&CHCQ23 have shown (Figure 9)higher compatibility compared with the other samples. This is inferred that due to the higher concentration of CQ components in CHCQ11&CHCQ23 samples, this improvement was possible. It is further understood that the surface coating has significantly improved the blood compatibility of the braided sutures

Figure 9.

Hemolytic activity of Flax braided sutures.

Conclusion

In the present work, the Flax fiber-based braided sutures were treated with chitosan/Cissus quadrangularis mixtures in different proportions. An 8-spindle braiding machine was utilized for the preparation of sutures with a 4 mm diameter. This braiding technique was aimed to improve the tensile strength of the suture and secure it after suturing, specifically for Anterior cruciate ligament surgery. The surface coating was given through the pad-batch technique. Four different kinds of suture samples were produced by changing the proportion of chitosan/Cissus quadrangularis. All the mechanical properties data were evaluated individually, and found that all test samples possessed sufficient tensile properties. CHCQ11&CHCQ23 have exhibited the best functional performance across the different suture samples. Samples CHCQ11&CHCQ23 showed increased cell growth through MTT on cell viability assay and higher compatibility of medical devices and blood. Similarly, Samples CHCQ11&CHCQ23 possessed higher antimicrobial and antifungal resistance than other suture samples due to its higher add on percentage of Cissus quadrangularis extract. The difference between the ultimate tensile stress among the samples was insignificant. The cell growth was found to be good for the samples that had a greater Cissus quadrangularis content. Since all the sutures were made through a braiding machine, it not only enhances the mechanical and elongation properties but also improves the structural integrity of the Sutures. Finally, these types of sutures are cost-effective and biodegradable due to the incorporation of natural extracts.