Abstract

Hyaluronic acid (HA) dermal fillers, derived from animal or non-animal sources, vary in physical and chemical properties, with numerous clinical trials assessing their effectiveness. This study examined the effects of Neuramis Volume Lidocaine on experimental animals over five durations (2, 4, 9, 10, and 13 weeks) via subcutaneous injections of 0.1 ml. Results showed a significant reduction in red blood cells, hemoglobin, and platelets after two weeks, which normalized by weeks 10 and 13. White blood cells and neutrophils significantly increased at two weeks but stabilized thereafter, while lymphocyte levels showed no significant long-term changes. Monocytes significantly decreased at two weeks but normalized by weeks 9-13. Scanning electron microscopy (SEM) revealed skin changes, including collagen bundle alterations and surface perforations. By week 10, the skin showed well-defined collagen structures and dense papillae, while earlier weeks exhibited more pronounced damage. These findings highlight HA fillers’ potential short-term physiological and histological impacts, which diminished over time.

Highlights:

1. Hyaluronic acid fillers vary in sources, properties, and clinical effectiveness.
2. Blood parameters altered early; histological changes observed, diminishing over time.
3. Fillers cause temporary physiological and histological issues, improving with duration.

Keywords: Hyaluronic acid, Histological study, Physiological study, Laboratory rats

Introduction

Hyaluronic acid (HA) is a prevalent dermal filler. It is a glycosaminoglycan polymer consisting of two disaccharide sugar molecules, including D-glucuronic acid and N-acetyl-glucosamine interconnected by glycosidic linkages. Hyaluronic acid (HA) comprises linear polymers with molecular weights ranging from 105 to 107 kilodaltons. The HA filler is distinguished by its capacity to bind and hold water molecules [1] ,Dermal fillers have received approval from many clinical medical and cosmetic trials [2].

Injectable dermal fillers possess several features, including efficacy, cost-effectiveness, safety, painlessness, biocompatibility, and tissue stabilization capability. The body should have a minimum response to the injected substance, which must be non-allergenic and stable at ambient temperature, Localized color alterations in the injection sites have been noted, resulting in heightened physiological changes, complications, and vascular blockage accompanied by discomfort. This leads to localized anemia characterized by dark blood coloration, maybe due to blood not traversing the capillaries as a result of pressure, accompanied by skin necrosis[3] .

The prevalent adverse effects may last for several days to one-year post-injection and encompass erythema, pruritus, edema, and nodules that may result in abscess development [4] .The most notable late effects are persistent capillary dilatation (telangiectasia) and granulomas in all fillers administered into soft tissues[5] .

The advancement and introduction of various sorts and formulations of HA dermal fillers necessitated the demonstration of their biodegradability as a precautionary measure for unforeseen circumstances utilizing hyaluronidase [6].

Methods

Dermal filler

The HA Filler, part of the NEU Neuramis Volume Lidocaine (NVL) range of Medytox Inc., Seoul, South Korea, was utilized. This dermal filler is a monophasic hyaluronic acid formulation, including a concentration of 20 mg/mL of hyaluronic acid and incorporating 0.3% lidocaine[7].

Animals

The present investigation was executed in April 2023 including male and female laboratory rats of the species Rattus norvegicus, namely Albino rats, aged 3 to 4 months, with an average weight ranging from 200 to 250 grams. They were housed in plastic cages at a temperature of 18-25 degrees Celsius and a relative humidity of 55 ± 15%, with provisions of water and food. A one-week acclimation period preceded the trial [8].

Experiment design

This investigation aimed to investigate the impact of the dermal filler Hyaluronic acid (HA) on hairless male and female laboratory rats. Each group was partitioned into five subgroups (A, B, C, D, E), with ten rats per subgroup. All groups were then administered the HA Filler dermal filler at a subcutaneous dosage of 0.1 mL. [9] .

Hematological measurements

The blood parameters were assessed post-blood withdrawal with the Auto Hematology Analyzer Genex Count-60 at the Al-Hayat Medical Laboratory in Iraq. The measurements comprised red blood cells (RBC), white blood cells (WBC), platelet count (PLT), and hemoglobin concentration (Hb) [10].

The differential count of white blood cells was determined by applying a drop of blood to a glass slide at a 45-degree angle until a uniformly thick smear was created. The specimen was subsequently stained with Leishman stain for 1-2 minutes, diluted with distilled water, and allowed to sit for 8-10 minutes. Subsequently, it was rinsed with running water and air-dried in preparation for testing. The sample was subsequently analyzed using a light microscope, and the proportions of monocytes, eosinophils, and lymphocytes were determined[11] .

Ultrastructural study

The research used skin samples sliced into 1 mm³ segments and preserved in a 2.5% Glutaraldehyde solution. The samples were subsequently rinsed with Phosphate Buffered Saline (PBS) and fixed in a 1% osmium tetroxide solution for 90 minutes. The samples were rinsed with distilled water twice for ten minutes each, followed by exposure to ascending concentrations of ethanol for 15 minutes. The samples were subsequently immersed in a pure propylene oxide solution for 30 minutes. The pure Araldite embedding media was utilized, and the sections were made with a thickness of 0.5 to 1 millimeter and dyed with a 1% Methylene blue solution[12]. The specimens were subsequently scanned with a scanning electron microscope.

Statistical analysis

Statistical analysis of all data was performed utilizing SPSS v.27 and Microsoft Excel 2019 for graphical representations, with data presented as mean ± SD (standard deviation). Analyses of changes were conducted utilizing one-way and multi-way ANOVA testing, with subsequent multiple comparisons between groups executed via Duncan’s post hoc test. The importance of differences was demonstrated at a probability level of (p<0.05) [13].

Result and Discussion

The effect of dermal fillers on Hematological standards

The current study's results indicated a significant reduction in red blood cell counts for both males and females treated for two weeks, relative to the control group over the same duration and to groups treated for 9, 10, and 13 weeks, at a probability level of p<0.05. Conversely, there was an insignificant increase in red blood cell counts compared to the group treated for four weeks, which itself exhibited a significant decrease when compared to the control group and the groups treated for 9, 10, and 13 weeks. The results indicated that the group treated for nine weeks exhibited a substantial reduction in red blood cell count compared to the control group over the same duration; however, no significant difference was observed when comparing the two groups treated for ten and thirteen weeks. Furthermore, when comparing the final groups (10, 13) throughout the course of weeks, no significant change was seen in the red blood cell count relative to the control groups during the same timeframe, as illustrated in Table 1.

The study results for both males and females indicated a significant decrease (p<0.05) in hemoglobin levels when comparing the two-week treatment group to the control group over the same duration, as well as to groups treated for 9, 10, and 13 weeks. However, no significant difference was observed when comparing to the group treated for four weeks. The study indicated that the latter group experienced a notable reduction in hemoglobin levels compared to the control group over the same duration, as well as in comparison to the groups treated for 9, 10, and 13 weeks. Specifically, the group treated for nine weeks demonstrated a significant decrease relative to the control group for the same timeframe, with no significant difference observed when compared to the groups treated for 10 and 13 weeks. Furthermore, there was no significant difference between the two groups treated for 13 and 10 weeks when assessed against their respective time controls or in comparison to each other, as detailed in Table 2.

The statistical analysis results for males and females indicated a significant decrease (p<0.05) in the platelet count rate when comparing the two-week treatment group with the control group over the same duration. In contrast, an insignificant decrease was noted when comparing this group with those treated for nine, ten, and thirteen weeks. Additionally, there was an insignificant increase when compared to the four-week treatment group, which itself exhibited an insignificant decrease relative to the groups treated for nine, ten, and thirteen weeks, alongside a significant decrease when compared to the control group for the same duration. The groups treated for nine, ten, and thirteen weeks did not demonstrate any significant differences when compared to the control groups for the corresponding periods, nor was there any significant difference observed among them, as detailed in Table 3.

The findings of the present study indicated a notable rise in the white blood cell count among both males and females in the treatment group over a two-week duration, in contrast to the control group for the same timeframe. Additionally, a significant increase was observed when compared to other groups across the time intervals of 13, 10, and 9 weeks, whereas no significant difference was noted with the treatment group at the 4-week mark. This group demonstrated a notable increase in comparison to the control over the same duration and relative to the other treatment groups during the specified time periods of 13, 10, and 9 weeks. However, no significant differences were observed among the treatment groups for these time periods at the probability level of p<0.05, as illustrated in Table 4.

The statistical analysis results of the current study for both males and females, as presented in Table (5), demonstrated a significant increase in the rate of neutrophils in the treatment group over a two-week period compared to the control group during the same timeframe. In contrast, there was an insignificant increase in the rate of neutrophils when comparing the two-week treatment group with those treated for 13, 10, and 9 weeks, and no significant difference was observed when compared to the treatment group for four weeks. The treatment group over four weeks exhibited a negligible increase in the rate of neutrophils in comparison to the groups treated for 13, 10, and 9 weeks. However, it demonstrated a significant increase relative to the control group during the same timeframe, as the rate of neutrophils in the control group was noted. The findings of the current study indicated no significant difference between the groups treated for 13, 10, and 9 weeks when compared to the control group over the same duration, with a probability level of p<0.05.

The study results for both males and females indicated a non-significant decrease in the lymphocyte rate when comparing the two-week treatment group with the control group over the same duration, as well as with the four-week treatment group. Additionally, there was no significant difference (p<0.05) in the lymphocyte rate when comparing it with groups treated for varying durations of 13, 10, and 9 weeks. The statistical results indicated a non-significant decrease in the lymphocyte rate when comparing the group treated for four weeks with the control group over the same duration. Additionally, there was no significant difference in the lymphocyte rate when comparing the groups treated for the durations of 13, 10, and 9 weeks. Furthermore, the results for the lymphocyte rate in females demonstrated no significant difference among the groups treated for 13, 10, and 9 weeks when compared with each other and with the control group for the corresponding time periods, as illustrated in Table 6.

The statistical analysis results of the current study in males indicated a significant decrease (p<0.05) in the rate of monocytes when comparing the treatment group over two weeks with the control group and the groups treated for durations of 13, 10, and 9 weeks. However, the results did not reveal a significant difference when comparing the two-week treatment group with the group treated for four weeks. A notable reduction in the monocyte rate was recorded for the group undergoing treatment for four weeks, in comparison to the control group over the same duration and to the groups treated for 13, 10, and 9 weeks. Conversely, the treatment groups of 13, 10, and 9 weeks did not exhibit any significant differences in monocyte rates among males when compared to one another and to the control group for their respective time periods, as detailed in Table 7.

*Significant differences at p-value <0.05. NS: non-significant. Multivariate ANOVA and compare between means by Duncan's post hoc test. each treatment groups 5 mice.

*Significant differences at p-value <0.05. NS: non-significant. Multivariate ANOVA and compare between means by Duncan's post hoc test. each treatment groups 5 mice.

*Significant differences at p-value <0.05. NS: non-significant. Multivariate ANOVA and compare between means by Duncan's post hoc test. each treatment groups 5 mice.

*Significant differences at p-value <0.05. NS: non-significant. Multivariate ANOVA and compare between means by Duncan's post hoc test. each treatment groups 5 mice.

*Significant differences at p-value <0.05. NS: non-significant. Multivariate ANOVA and compare between means by Duncan's post hoc test. each treatment groups 5 mice.

*Significant differences at p-value <0.05. NS: non-significant. Multivariate ANOVA and compare between means by Duncan's post hoc test. each treatment groups 5 mice.

*Significant differences at p-value <0.05. NS: non-significant. Multivariate ANOVA and compare between means by Duncan's post hoc test. each treatment groups 5 mice.

The effect of dermal fillers on skin tissue

The results of the scanning electron microscope (SEM) examination of the skin tissue in male and female laboratory rats within the control group indicated an irregular and rough dermal surface, accompanied by small holes at the base of the dermal papillae in the epidermis layer, as depicted in Figure (1). Furthermore, the treated group, comprising both males and females who received dermal filler Hyaluronic acid for two weeks following the injection, displayed folds and holes on the textured dermal surface. Thin collagen bundles were observed on the papillary surface, accompanied by thin and elongated hair, as depicted in Figure (2). The group treated with dermal filler for four weeks exhibited rough skin areas with noticeable folds and clear holes, alongside smoother regions characterized by thin collagen, as illustrated in Figure 3. In contrast, the group treated for nine weeks post-injection demonstrated the formation of thick, folded collagen bundles with holes in the dermal papillae, as well as the presence of dense, long hairs, as depicted in Figure 4. The group that underwent treatment for ten weeks exhibited the presence of papillae. Distinct dermal papillae exhibit thick and dense collagen, alongside openings that manifest in the grooves between these papillae and elongated hair, as illustrated in Figure (5). Furthermore, the treatment group, observed over a period of 13 weeks post-injection, displays a smooth collagenic surface with well-defined trabecular dermal papillae. Notably, wide openings and robust, smooth collagen bundles are evident, accompanied by long and thick hair, as depicted in Figure (6).

Figure 1.Area of rat skin. (control group). Note irregular and rough dermal surface with invaginations (I) and there are the small holes visible at the origin of dermal papillae demonstrating the presence of free nervous fibers entering the epidermal layer (H).

Figure 2.Area of rat skin. (2 week injection). Higher magnification, showing rough dermal surface with presence of invaginations (I) and holes (H) in the dermal surface. Also there are thin collagen bundles (C) and thin and long hairs (HA).

Figure 3.Area of rat skin. (4 week injection). Note rough area of skin and few invaginations (I) with holes (H). Also there are other areas are smooth (S) in the dermal surface with thin collagen (C).

Figure 4.Area of rat skin. (9 week injection). Higher magnification of the previous section. There are dermal papillae which contains holes and thick collagen bundles (C).

Figure 5.Area of rat skin. (10 week injection). There are marked dermal papillae (P) with dense and thick collagen (C) and note the holes (H) which opened in the grooves formed between these papillae. Long hair may be seen (HA).

Figure 6.Area of rat skin. (13 week injection). There are the smooth collagenic surface (C) with marked trabecular dermal papillae (P) with wide holes (H). Long and dense hair may be seen (HA).

Discussion

The findings of the present study indicated variations in the counts of red blood cells and hemoglobin among the groups administered hyaluronic acid dermal filler for durations of 2 and 4 weeks, in comparison to one another and to the control groups. Conversely, the groups observed over the periods of 9, 10, and 13 weeks did not exhibit any significant differences when compared to each other. The ability of HA filler to penetrate tissues can lead to an increase in the blood sedimentation rate, which may occur due to simple infections or anemia. This aligns with the findings [14], who noted that HA filler with a molecular weight of 35 kDa demonstrates significant tissue penetration capabilities, along with anti-inflammatory properties, leading to erythrocyte aggregation and increased sedimentation rates due to CD44's binding to the cell surface [15]. Conversely,[16] indicated that HA, characterized by a very high molecular weight, exhibits limited tissue penetration, resulting in diminished effects, which accounts for the transient nature of dermal fillers as observed in our study.

The findings of our research align with those of [17], which indicated that biosynthesis in the majority of human cells can produce HA during specific phases of the cell cycle. Additionally, it was noted that the half-life of HA in the bloodstream is approximately 24 hours[18], and that the equilibrium between HA synthesis and degradation is crucial for regulatory functions within the human body [19]. It was observed that activated fibroblasts can generate significant quantities of HA, while a smaller fraction is transported into the bloodstream and subsequently degraded by liver endothelial cells[20]. This elucidates the transfer of HA into the blood in our study and its transient impact on blood parameters.

The total platelet count demonstrated a reduction in the groups treated for 4.2 weeks in comparison to the control and other treated groups. In contrast, no significant differences were observed among the groups treated for durations of 13, 10, and 9 weeks when compared to each other and the control. This phenomenon can be attributed to the ability of HA to diminish wounds and expedite the healing process, resulting in a decrease in the platelet rate shortly after injection. The absence of an increase in the incidence of blood clots may be attributed to the role of HA fillers in various physiological processes, including wound healing and blood clotting, as confirmed by [21]. HA fillers promote the activity of fibroblasts, resulting in the deposition of type I and type III collagen [22]. Furthermore, HA has the capability to interact within the vessels with fibrinogen, thereby enhancing the formation of fibrin clots induced by thrombin [23], which subsequently promotes tissue healing. The rationale behind the reestablishment of the criteria for the increase in platelet count during later periods may be linked to the impact of dermal fillers on tissues, resulting in tissue damage, necrosis, and rupture of blood vessels, consequently leading to bleeding. This necessitates the enhancement of platelet levels to mitigate the risk of blood loss.

The findings of the current study demonstrated an increase in the total count of white blood cells and neutrophils in the treatment groups for 4 and 2 weeks, in comparison to the control groups and those treated for other durations of 13, 10, and 9 weeks. The lymphocyte levels showed no significant differences across all treatment periods. Additionally, a decrease in monocyte levels was observed in the groups treated for 4 and 2 weeks, with no significant differences noted among the groups treated for the longer durations of 13, 10, and 9 weeks. and the increase in the number of white blood cells can be explained by the occurrence of infections resulting from the injection of foreign material into the body, as the study [24],showed that the level of WBC is proportional to wound inflammation, as it was observed that WBC values increased after wounds and reached their maximum on day (7) and then decreased to a constant value, and when comparing the groups, the value in the control group was higher than the group treated with HA filling in each time period, and the differences in WBC values between the HA group and the control group were large at (11,9,7) days and with a significant difference (P < 0.05), proving that the inflammatory state of the HA group was less severe than that of the control group at (11) days after the wound operation, and the WBC results showed that the animals in the HA group had the lowest values during the first (17) days, which implied that they had less severe inflammation.

The analysis of skin tissue using scanning electron microscopy (SEM) in the group treated with hyaluronic acid dermal filler for two weeks post-injection revealed the presence of collagen bundles on the papillary surface accompanied by fine hair. The group treated for four weeks exhibited a skin surface that was both rough and smooth, characterized by folds, clear holes, and thick, folded collagen bundles, along with the presence of dense hair. The group treated for 10 weeks exhibited clear dermal papillae accompanied by thick collagen, along with openings in the papillae grooves of the skin where long hair was visible. In contrast, the group treated for 13 weeks post-injection displayed a smooth collagenic surface with distinct trabecular spongy dermal papillae, characterized by wide openings and thick collagen bundles, along with long and thick hair.

The alterations can be elucidated through the study conducted by [25], which demonstrated the impact of hyaluronic acid on the thickness, hardness, and protection of hair. SEM examination revealed that hyaluronic acid dermal filler enhances hair moisture by improving mechanical properties, as well as increasing hair thickness through a layer formed by the filler on the hair surface. This layer is composed of hydrogen bonds between the side groups of keratin in the hair and the functional groups of the hyaluronic acid filler. This demonstrates the absorption of the filler on the hair surface, resulting in a smooth and soft texture. The SEM examination confirmed that hair scales are more separated in the absence of dermal filler. The presence of HA acid enhances hair smoothness and scale separation, while it does not influence cell growth or repair within the hair. However, it temporarily improves the external appearance of damaged hair by reducing surface free energy and roughness.

Our study's results are consistent with those of [21], who observed that hair-derived hydrogels had a white appearance and good fluidity and viscosity. Because of these characteristics, they can be injected into soft tissues to increase the volume of the epidermis' outermost layer. The original tissue was retained by the filler particles in a variety of forms, including bundles and fibers, according to an analysis using electron microscopy (SEM). Because of its structural integrity, the filler was able to remain effective and retain its properties in the subcutaneous layer for a long time. For 48 weeks after the filler injection, there was no outward rejection or inflammation. The study's microscopic analysis [26] verified that wounds treated with HA showed thicker layers of the epidermis and dermis.

We found that keratin fibers are crucial for epithelial cell development and hair follicle regeneration because they stimulate cell proliferation and adhesion more effectively than fibers containing hyaluronic acid [27]. Our findings are in line with that study. The fiber sheets containing HA or keratin were wider than the control group, according to scanning electron microscopy. This is because topical fillers penetrate the skin through lipid channels between keratinocytes, hair follicles, or sweat glands. The HA filler reduces hydrophobicity because it has a high capacity to absorb water, provide the skin with the right amount of moisture, and give the entire skin tissue elasticity. However, some of these fibers were broken and deformed in comparison to keratin fibers [28].

Conclusion

The findings of this study indicate that the use of hyaluronic acid dermal fillers induces varying hematological and histological effects depending on the duration of treatment. Notably, short-term exposure (two weeks) resulted in significant reductions in red blood cell counts, hemoglobin levels, and platelet counts, alongside increases in white blood cells and neutrophils, suggesting an acute physiological response. These effects diminished or became statistically insignificant over longer treatment durations (9, 10, and 13 weeks), indicating a potential adaptation or recovery over time. The histological analysis revealed alterations in skin collagen and dermal structure, with short-term treatments showing less favorable outcomes compared to the improvements observed in the later stages of the study. These findings underscore the importance of monitoring hematological parameters and histological changes when using dermal fillers, especially in the initial phases of treatment. The study highlights the need for further research to explore the underlying mechanisms of these responses, evaluate their clinical relevance, and investigate potential long-term implications in human models to ensure the safety and efficacy of hyaluronic acid-based fillers.

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