Prior hemostasis evaluation, both the chemical structure and morphology of the used hemostatic materials were analyzed since these parameters are crucial for the hemostasis efficiency.
Chemical Composition
The results of ATR-FTIR spectroscopy spectra comparing the chemical structure of the used hemostats are shown in Fig. 1. The ATR-FTIR spectrum of the controlled ORC fibril (blue line) displayed a small broad absorption band of approx. 3600 to 3000 cm−1 that belonged to the stretching vibration of the hydroxyl –OH groups. This broad band was more visible in CMC samples, while the largest was represented by the CMC Na sponge (pink line). Interestingly, no band was visible in this region in the PP/Vis needle-punched sample (black line). However, this sample did show the largest bands at 2918 and 2848 cm-1, confirming the C–H stretching vibration of the –CH2 polypropylene groups. The band that resulted from the ring stretching of glucose appeared at 1611 cm−1 at CMC samples. The bands at 1434 and 1340 cm−1 included –CH2 scissoring and C–OH bending vibrations, respectively. The band at 1160 cm−1 was assigned to the asymmetric vibration of the glycosidic bonds in cellulose molecules. Very strong absorption bands at 1058 and 1028 cm−1 were attributed to skeletal vibrations involving the stretching of C–O and C–C bonds, respectively, that are attached to the glucose rings. The weak bands at approximately 720 cm−1 were due to ring stretching and ring deformation of α-D-(1–4) and α-D-(1–6) linkages (El-Sakhawy et al. 2000; Fukuzumi et al. 2010; Wang et al. 2005). To conclude, the highest variant was the PP/Vis needle-punched sample affected by the polypropylene network. The largest number of both hydroxyl and carboxyl groups showed samples CMC (CMC Na sponge - pink line and CMC Na/Coll sponge – orange line), while CMC H spunlace (violet line) was the most chemically similar to control ORC fibril (blue line).
Morphology of Hemostats
In addition to the chemical composition, the size and shape are other important factors that determine the properties of functional materials. As shown SEM images in Fig. 2, the structure of used hemostatic materials was very different. Samples a) (CMC Na sponge) and b) (CMC Na/coll sponge) were porous foams prepared by freeze-drying. Interconnected pores permitted fast liquid absorption. Very similar was the structure of knitted fabrics of sample c) (CMC Na knit) and d) (CMC H knit) - CMC H knit was actually acidic form made of CMC Na knit, where sample c) was scanned from the top and sample d) from the bottom. Other samples e) (PP/Vis n-punched), f) (CMC Na n-punched) g) (CMC H spunlace) and h) (ORC fibril) yielded very similar fibrilar morphology, where just sample e) had a significant PP network on the surface of viscose, probably due to better mechanical properties and manipulation. Porosity of the materials was determined and the results are shown in Table 2. The highest porosity was found out for CMC Na/Coll sponge followed by CMC Na knit. Other materials revealed lower porosity. Interestingly, the absolutely lowest porosity of all materials was calculated at CMC Na sponge. Absorption capacity was determined as well. Results are shown in Fig. 3. Sponges followed by knits had the lowest free swell absorptive capacity.
Table 2 Porosity of hemostatic materials
Sample
|
Porosity (%)
|
CMC Na sponge
|
7.6
|
CMC Na/Coll sponge
|
72.8
|
CMC Na knit
|
49.9
|
CMC H knit
|
22.1
|
PP/Vis needle-punched
|
11.8
|
CMC Na needle-punched
|
27.6
|
CMC H spunlace
|
20.3
|
ORC fibril
|
20.6
|
Time to Hemostasis
The bleeding was completely stopped before the peritoneum was closed again. No animal bled to death immediately after the surgery or during the convalescence period. We recorded significantly shorter time to hemostasis in the CMC Na sponge (6.1 ± 3.7 s), the CMC Na/Coll sponge (6.8 ± 3.5 s) and the CMC Na knit (9.5 ± 4.2 s) compared to the control ORC fibril (34.6 ± 9.5 s). Both sponges also reached significantly less time than the CMC Na needle-punched (28.5 ± 7.8 s), the CMC H spunlace (28.6 ± 9.5 s) and the PP/Vis needle-punched (23.8 ± 7.3 s). The CMC Na knit proved to have significantly better hemostatic effect compared to the CMC Na needle-punched and the CMC H spunlace. The results with the observed statistically significant differences are summarized in the graph in Fig. 4.
Necropsy and Macroscopic Findings
One half of the animals from each group were evaluated after 3 days. The remaining half was evaluated after 30 days.
In the CMC Na sponge, the material was completely absorbed in one case. Other cases revealed remnants of material with traces of blood in one rat. After 30 days, the material was completely absorbed. There was a pale-coloured kidney in one case and a substantial amount of fat around the intestine.
In the CMC Na/Coll sponge, the remaining material with traces of blood was observed on the wound at the 3-day evaluation. The 30-day assessment confirmed the bioresorbability of the material.
In the CMC Na knit, the material remained attached to the wound in one case. Other cases revealed bioresobability after 3 days. The kidney had a pale appearance in 3 rats. After 30 days, the kidneys looked healthy and there was no material remaining.
In the CMC H knit, the material transformed into a gel and remained on the wound after 3 days. After 30 days, the material was completely absorbed.
In the PP/Vis needle-punched, the material was stuck to the wound at the 3-day evaluation. The kidney had a pale look in one case. Although after 30 days the material remained on the wound, there were no signs of granulomatous reaction.
In the CMC Na needle-punched, the used material was stuck to the wound at both evaluation periods. Granulomatous reaction prevailed in most cases of the 30-day evaluation. There was a significant abscessing inflammation, particularly in one rat in this assessment.
In the CMC H spunlace, the material was still on the wound at the 3-day evaluation. There was one pale kidney and a trace amount of ascites. After 30 days, the material remained in the rat’s body. There were signs of granulomatous reaction in most cases. An icteral colour of fat around the kidney was registered in one rat.
In the ORC fibril, the remaining material was observed on the wound after 3 days. The tested material adhered to the wound and revealed traces of blood. The manufacturer has guaranteed the bioresorbability after 7-14 days. Therefore, the assessment after 30 days was a decisive aspect for us and it confirmed the bioresorbability of the material. One pale kidney was detected in a tested rat. The finding may be attributed to a more extensive blood loss. The remaining kidneys retained a healthy appearance.
Histopathological Evaluation
All monitored histopathological features are summarized in Table 3. The total destruction score revealed a direct influence on the healing tissue. The group with the lowest number was thus considered the most suitable material of this study.
Table 3 Histopathological evaluation after 3(a) and 30(b) days. Followed parameters: D, tissue destruction; F, extent of fibroproduction; R, reaction of the surrounding tissue; I, inflammatory infiltration
|
|
Evaluated parameters
|
|
|
Resorbability
|
D
|
F
|
R
|
I
|
Destruction score
|
CMC Na spongea
|
no
|
1,1,1,0.5,1
|
1,0.5,0.5,0.5,0.5
|
1,0.5,0.5,0.5,0.5
|
1,0.5,0.5,0.5,1
|
14
|
CMC Na/Coll spongea
|
no
|
1.5,1,1.5,1,1.5
|
1,0.5,1,1,1
|
1.5,1.5,1.5,1,0.5
|
1.5,1,1.5,1,1
|
23
|
CMC Na knita
|
yes
|
0.5,1.5,1.5,1.5,1
|
0.5,1,1,1,1
|
0.5,1.5,1.5,1,1
|
0.5,1,1,1,1
|
20.5
|
CMC H knita
|
no
|
1,1,1,1,0.5
|
1,1,1,1,1
|
1,1,1,1,1
|
2,1.5,1.5,1,0.5
|
21
|
PP/Vis needle-puncheda
|
no
|
1,1,1,1,1
|
1,0,0,1,1
|
1,2,1,1,1
|
1,1,1,1,2
|
20
|
CMC Na needle-puncheda
|
no
|
1,1,1,2,1
|
1,1,1,1,1
|
1,2,2,2,1
|
1,1,1,1,1
|
24
|
CMC H spunlacea
|
no
|
2,1,1,1,2
|
1,1,1,1,0
|
2,1,2,2,2
|
2,1,1,1,1
|
26
|
ORC fibrila
|
no
|
2,1,2,2,1
|
1,1,1,1,1
|
1,1,1,1,2
|
1,1,1,1,2
|
25
|
|
|
|
|
|
|
|
CMC Na spongeb
|
yes
|
1,0.5,0.5,0.5,0.5
|
0.5,0.5,0.5,0.5,0
|
1,0.5,0.5,0.5,0.5
|
1,0.5,0.5,0.5,0.5
|
11
|
CMC Na/Coll spongeb
|
yes
|
0.5,1,0.5,1,1
|
0.5,1,0.5,1,1
|
0.5,0.5,0,1,1
|
0.5,1.5,0.5,2,1.5
|
17
|
CMC Na knitb
|
yes
|
1,0.5,1.5,1,0.5
|
1,1,0.5,1.5, 0.5
|
0.5,0.5,1,1,0
|
0.5,0.5,2,2,0
|
17
|
CMC H knitb
|
yes
|
0.5,0.5,0.5,0.5,1
|
0.5,0.5,0.5, 0.5,1
|
0.5,1,0.5,0.5,1.5
|
1,1,0.5,1,2
|
15.5
|
PP/Vis needle-punchedb
|
no
|
2,1,1,0,1
|
1,0,1,0,0
|
1,1,1,1,1
|
1,0,1,0,1
|
15
|
CMC Na needle-punchedb
|
no
|
1,2,0,1,1
|
2,2,2,2,2
|
1,2,1,1,1
|
2,2,0,2,2
|
29
|
CMC H spunlaceb
|
no
|
1,2,2,1,1
|
2,2,2,2,2
|
1,1,2,2,1
|
2,2,2,2,2
|
34
|
ORC fibrilb
|
yes
|
1,2,1,1,1
|
1,2,1,1,2
|
1,2,2,1,1
|
1,2,2,2,2
|
29
|
In the CMC Na sponge samples revealed small clear zones of inflammation and necrosis after 3 days (Fig. 5a). The destruction score was 14. A relatively favorable healing reaction appeared after 30 days. Almost clear cutting line with only a slight inflammatory reaction still occurred (Fig. 6a). The destruction score was 11. The CMC Na sponge had the best result considering both destruction scores.
In the CMC Na/Coll sponge samples showed massive inflammation and presence of necrosis after 3 days (Fig. 5b). The total destruction score was 23 after 3 days and 17 after 30 days. The evaluation after 30 days revealed the presence of granulomas, bleeding, and a clear area of inflammation (Fig. 6b).
In the CMC Na knit, samples were characterized by necrotic tissue. Renal tubules with massive inflammation around appeared in the preparations (Fig. 5c). The destruction score was 20.5 after 3 days. This score decreased after 30 days to number 17. After a longer period, the parenchyma showed very significant inflammation and the presence of granulomas (Fig. 6c).
The CMC H knit material caused complete destruction of the tissue by necrosis after 3 days. There was massive inflammation and signs of bleeding (Fig. 5d). The total destruction score was 21. The number decreased after 30 days to 15.5. A relatively narrow area of inflammation appeared after a longer period (Fig. 6d).
In the PP/Vis needle-punched, samples showed a smooth cutting line with the hematoma after 3 days. There were protein cylinders with significant reaction (Fig. 5e). The total destruction score was 20. The score decreased after 30 days to number 15. There was just a slight inflammatory reaction and subsequent scarring (Fig. 6e).
In the CMC Na needle-punched, samples were characterized by damaged tissue with protein cylinders and the clear sign of fibroproduction after 3 days (Fig. 5f). There was also the presence of a hematoma and thrombotic blood vessels in one case. The total destruction score was 24. The evaluation after 30 days revealed the rest of the material and inflammatory reaction with significant scarring (Fig. 6f). Finally, the total destruction score was 29.
In the CMC H spunlace, samples were characterized by fibroproduction after 3 days. The assessment revealed necrosis and inflammatory reaction around (Fig. 5g). The total destruction score was 26. The parenchyma was quite similar to the CMC Na needle-punched after 30 days. There was the rest of the material with significant formation of scar tissue (Fig. 6g). The total destruction score was 34. This material showed the worst effect on the tissue at all
After 3 days, in the ORC fibril, necrosis and dispersed inflammatory cells were revealed (Fig. 5h). The total destruction score was 25. After 30 days, the parenchyma showed signs of granulomatous reaction around the foreign material and chronic inflammatory infiltration (Fig. 6h). The total destruction score increased to 29.