Experimental investigation of biostimulatory effects after polydioxanone thread insertion in a pig model

Polydioxanone (PDO) threads have been widely used to tighten and lift the facial soft tissue.


| INTRODUC TI ON
Thread-lifting plastic surgery techniques are favored due to their relative safety, ease of procedure and effectiveness, and low risk of side effects. 1,2Recently, absorbable threads have been developed as an alternative to non-absorbable cogged threads, which are associated with rejection and are not conducive to adjustment. 3 These absorbable threads mechanically lift the skin immediately after implantation, 4 and do so for approximately 6 months, when the threads degrade through hydrolysis. 5Simultaneously, the implanted threads can stimulate new collagen formation in the tissue, 6 which can last for 2-3 months after the degradation of the threads. 7At present, the most popular thread material is polydioxanone (PDO), a synthetic monofilamentous polymer of multiple repeating ether-ester units, which lasts up to an average of 6 months after hydrolysis and disappears completely within the tissue, 8 which has low tissue reactivity and extended tensile strength after embedding. 8,9PDO threads cause specific changes of surrounding tissues that works by triggering fibroblasts to produce more collagen, 10,11 which affects downstream signaling molecules 7 and causes skin-rejuvenating effects. 12The nylon thread, as a traditional non-absorbable suture, has been widely used in clinical practice, and there are records of nylon thread used in early thread-lifting.Non-absorbable sutures show an initial tensile profile that is superior to absorbable material. 13Nevertheless, the thread can become palpable or cause unexpected foreign body reaction and chronic inflammation in some patients because of long-term implantation of non-absorbable sutures. 14,15Nylon threads and poly lactic-co-glycolic acid (PGLA) are also used and are NON-absorbable and absorbable, respectively.PGLA thread is a commonly used type of absorbable suture in clinical practice, containing 90% poly glycolic acid and 10% poly lactic acid, which causes a stronger inflammatory response and higher collagen production than PDO, yet PDO is a more popular material in facial rejuvenation. 16In tissues that are similar to human facial soft tissue, such as pig adipose tissue, it is unclear whether different threads trigger different inflammatory responses involved in collagen regeneration.Although several studies have compared the shape and mechanical effects of different threads such as Cog, monofilament, and multifilament, 10,17,18 few studies focus on the integrity and degradation of threads or collagen production.Therefore, we aimed to investigate the histological and collagen changes at 1, 4, 12, 24, and 48 weeks after implantation of PDO, PGLA, and nylon threads to evaluate collagen production and different degradation processes using a pig model.

| Thread
The absorbable PDO thread used in this study was manufactured by IMEIK Technology Development Co., Ltd (Beijing, China).The nylon and absorbable poly (glycolic-co-lactic acid) (PGLA) threads were acquired from Shandong Haidike Medical Products Co., Ltd.(Shandong, China) and Samyang Biopharmaceuticals Corp. (Gyeonggi-do, Korea), respectively.The PDO and nylon threads had monofilament thread, whereas the PGLA thread had a multiply braided thread.All threads were 75 cm and USP 2-0 non-barbed.

| Animals
Five female Bama miniature pigs aged 12 months (weight 25-30 kg) were purchased from Beijing Kuibushichuang Biotechnology Co., Ltd (Beijing, China).The pigs were kept under optimal conditions at a temperature of 23 ± 3°C, a relative humidity of 40%-70%, and a 12 h light/12 h dark cycle and fed with food and water ad libitum.After 1 week of acclimatization to the environment, the pigs were numbered and randomly divided into five groups by lottery.

| Experimental process
After the administration of Zoletil anesthesia (VIRBAC; Carros, France) via intramuscular injection, inhalation anesthesia was administered in combination with isoflurane (Jiangsu H.F.Q Biotechnology Co., Ltd; Jiangsu, China) in a sterile surgical room.We shaved the pigs and disinfected the dorsal skin with alcohol.In the dorsal skin, three threads of 75 cm lengths (PDO, PGLA, and nylon, Figure 1A) were inserted into the subcutaneous layer using an 18 G needle parallel to the skin.We ensured that the length of the folded line was at least 5 cm (Figure 1B).The insertion site of each thread was tattooed on the skin to facilitate pathological examination.The same procedure was performed in five Bama miniature pigs.Afterwards, the thread and surrounding subcutaneous tissue were biopsied under Zoletil anesthesia at five time points: 1, 4, 12, 24, and 48 weeks after thread insertion.We performed morphological evaluation on these tissues (Figure 1C).

| Tissue morphological observation
The tissue was collected and fixed in 10% neutral buffered formalin for paraffin embedding.It was microtomed vertically to the longitudinal axis of the thread at 4 μm thickness.The samples were stained with hematoxylin and eosin and Masson trichrome staining for light microscopic observations.

| After 1 week
The PGLA thread became loose between the threads at the experimental site, whereas the other two types of threads did not change significantly.A small amount of collagen fiber, eosinophils, and lymphocytes were visible near the PDO thread insertion site; additionally, visible bridging fibrous tissue could be seen at some sites.Severe tissue reaction could be seen around loose PGLA lines, and large quantities of irregular collagen fiber, eosinophils, and lymphocytes distribution were seen around the thread.Although the reaction around the nylon thread was similar to that around the PDO thread, the amount of collagen was much smaller than that in the PDO thread (Figure 2).After Masson trichrome staining, newly formed rich collagen fibers (stained light blue) were observed near the thread insertion site, which were loose in the PDO and nylon threads and more compact around the PGLA thread (Figure 3).

| After 4 weeks
The purple color of the PDO thread became transparent at the experimental site, and the structure of the PGLA thread disintegrated, with no change observed in the nylon thread.Abundant loose collagen fiber, eosinophils, and lymphocytes were visible inside the newly formed granulation tissue near the PDO thread insertion site.Two circular empty spaces were observed in the PGLA recipient area; this appearance was a result of half of the thread being inserted inside the needle and the other half exposed outside of the needle.The collagen fibers, eosinophils, and lymphocytes were only found within the loose residual material.The reaction around nylon thread was similar to that of PDO thread; however, the amount of collagen was much smaller than that of the PDO thread (Figure 2).
The superficial fascia layer thickened to some extent when the PDO thread was embedded around it (Figure 4).The presence of rich collagen fibers was similar for all three threads to Week 1.The PDO thread produced more collagen, which was clearly distinguishable from lymphocytes (Figure 3).

| After 12 weeks
The PDO thread was still transparent, the PGLA thread was largely invisible, and the nylon thread remained unchanged.At 12 weeks, many loose collagen fibers were found near the insertion site of the PDO thread, and eosinophils and lymphocytes were decreased when compared to the 4-week point.In the circular cavity of the PGLA thread, no significant material residue could be seen, and collagen fibers, eosinophils, and lymphocytes were significantly reduced at 12 weeks when compared to the 4-week point.The reaction around the nylon thread was similar to that of the 4-week point (Figure 2).Bridging of fibrous tissue and thickening of superficial fascia layer were also observed at this point around the PDO thread (Figure 4).After Masson's trichromatic staining, abundant newly formed collagen fibers could be seen near the PDO thread site (stained light blue).The number of collagen fibers was small, and the structures were loose near the PGLA and nylon thread sites (Figure 3).

| After 24 weeks
The PDO and PGLA threads were structurally disintegrated, while the nylon thread remained unchanged.Collagen fibers, eosinophils, and lymphocytes were seen in loose residues at the PDO thread insertion site, and the growth direction of collagen was directed to the superficial fascia layer.Some collagen fibers could be seen in the implanted area of the PGLA thread.There was slight change in the reaction around the nylon thread at one point (Figure 2).After Masson's trichromatic staining, abundant newly formed tight collagen fibers (stained light blue) were visible at the PDO thread site, and collagen could also be observed at the PGLA thread, while loose collagen tissues were still observed near the nylon thread (Figure 3).

| After 48 weeks
The PDO and PGLA threads were not visible, while the nylon thread remained unchanged.Some collagen fibers could still be seen in the implantation area of the PDO thread, while the implantation site of the PGLA thread was no different from normal tissue.The reaction around the nylon thread remained unchanged; however, the reaction area seemed to be smaller than 24 weeks (Figure 2).After Masson's  trichromatic staining, collagen (stained light blue) could also be observed at the PDO thread site, while loose collagen tissue was still visible near the nylon thread (Figure 3).

| DISCUSS ION
Our results showed that inflammation and collagen production in the PDO threads was mild, with peak collagen production at 12 weeks and collagen fibers persisting until 48 weeks.PDO thread began to degrade at 24 weeks and was completely invisible at 48 weeks, which was similar to the results of previous studies. 7,11,19We observed that the PGLA thread lost its integrity after 4 weeks and was almost degraded at 12 weeks, which was much shorter than the degradation time of the PDO thread.
This finding was consistent with results of previous studies. 20,21e inflammatory response caused by thread insertion is a key prerequisite for collagen production by fibroblasts after microparticle encapsulation.We found that each thread implantation induced a different intensity of inflammatory response, which correlates with the findings of another study. 17In the degradation process of the PDO thread after 24 weeks, the inflammatory reaction was relatively stable and mild, which has been previously shown. 7,11,19For PDO threads, the inflammatory response peaked at 4 weeks, and no dramatic changes were observed during the experiment.In contrast, the PGLA thread showed a strong inflammatory response in the early stages (before 4 weeks).We believe that the strong inflammatory response of the PGLA thread may be related to its loose structure and rapid degradation time, both factors that increase the contact area between the implant and the tissue.Large-scale inflammatory reactions may lead to foreign body granuloma, a severe side effect of thread-lifting.We found that the level of collagen formation for the PDO thread throughout 48 weeks was mild and was similar to that in previous studies. 11,23Contrastingly, with the strong inflammatory reaction of PGLA thread at the early stage, a large amount of irregular collagen appeared around the recipient area, which would likely be metabolized within a few weeks after thread degradation.The nylon thread showed a small amount of chronic inflammation throughout the implant cycle.Although the stimulation was long-lasting and sustained, there was no collagen accumulation.This may be due to the body metabolizing the collagen. 24terestingly, we also observed the fibrous bridging effect of the PDO thread in this experiment.The collagen fibers branched out from the thread insertion site toward the dermis and the fascia in a spider web shape.Furthermore, we observed the fibrous bridging effect between closely spaced thread.Previous research has shown that this bridging action can persist for more than 48 weeks. 11Similar to our findings, other research has found that the insertion of PDO thread around fibrous connective tissue increases the tissue thickness and joins pre-existing fibrous connective tissue, which stimulates soft tissue reinforcement through merging. 11In addition, we observed regular growth of new collagen toward the existing fibrous connective tissue, which may be caused by the fibrous bridging effect and thickening of fascia tissue.However, these formations were not common and most of them occur between two adjacent implant lines or around the existing fiber tissue.We speculate that this is related to the signal transmission between cells, where too far a distance leads to the failure of signal transmission.
Overall, compared to the PGLA and nylon threads, the PDO thread produced milder inflammation, less irritation of the skin, comparable collagen production, obvious bridging fibrous tissue capabilities, and more thickening of the superficial fascia.In clinical practice, long-term studies have also demonstrated the efficacy of PDO thread in achieving notable facial rejuvenation outcomes extending up to a duration of 2 years, and the synergistic impact of combining with adjunctive procedures such as Botox and platelet-rich plasma (PRP) has been found to significantly enhance these outcomes. 25Thus, we found similar evidence to previously published research that PDO threads are a safe and effective thread-lifting material for the treatment of aging skin and may be more suitable for clinical application.
This study had some limitations: the animal sample size was small and histological differences identified by staining may not reflect realistic changes.Future research into quantifying the changes, rather than describing qualitatively as we have done here, is recommended.Finally, we did not investigate the effect of different insertion techniques, which should be the focus of future research.

F I G U R E 1
Threads style and placement mode.(A) Photograph of polydioxanone (left), poly glycolic-co-lactic acid (middle) and nylon thread (right).(B) Schematic image of implant location.(C) Experimental design of the study.| 661 WANG et al.

F I G U R E 2
Hematoxylin and eosin stain of different thread inserted tissues.Formation of the cross-section (triangle) and the vertical section (quadrate) of the threads are shown.At 24 weeks, collagen was still visible at the site of poly glycolic-co-lactic acid thread implantation (circular), but at 48 weeks it was no different from normal skin (original magnification ×100; scale bar: 200 μm).

F I G U R E 3
Histological response to different threads after insertion based on Masson trichrome staining.Formation of the cross-section (triangle) of the threads and collagen (pentagram) around the thread can be seen.At 24 weeks, collagen was still visible at the site of poly glycolic-co-lactic acid thread implantation (circular), but at 48 weeks it was no different from normal skin (original magnification ×100; scale bar: 200 μm).

F I G U R E 4
Histological evaluation of tissue formation.(A) Bridging fibrous tissue (green triangle) between the adjacent thread is noted in 1 week (left) and 12 weeks (right) for the polydioxanone thread.(B) Newly formed collagen fibers (red triangle) have connected and increased with superficial fascia (blue square) at 4 (left) and 12 weeks (right) after polydioxanone thread insertion.(C) Newly formed collagen fibers linked to the superficial fascia (red arrow: indicate the growth direction of collagen) in 24 weeks for the PDO thread are shown.(hematoxylin and eosin; A and B original magnification ×50, scale bar: 200 μm; C original magnification ×20, scale bar: 500 μm).