Prospective comparative clinical study: Efficacy evaluation of collagen combined with hyaluronic acid injections for tear trough deformity

Tear trough filling is a popular facial rejuvenation procedure, and hyaluronic acid is typically used as the filler of choice. However, Tyndall's phenomenon, a common complication following hyaluronic acid injection, can occur, leading to skin discoloration of the lower eyelid.


| INTRODUC TI ON
2][3] However, factors such as wrinkles, tear trough deformities, and tissue structure deterioration may occur in the periorbital region. 4Tear trough is a concave deformity of the lower lid area resulting from changes in the periorbital tissue structure.The medial groove between the eyelid and cheek is often referred to as the nasojugal groove or tear trough, while the lateral groove is known as the palpebromalar groove. 5Multiple factors contribute to tear trough formation, including laxity of the lower eyelid skin, orbicularis oculi muscle, orbital septum, herniation of the lower eyelid fat, and alterations in bony structures. 6Additionally, hyperpigmentation of the lower eyelids or changes in illumination can accentuate the visibility of the tear trough.
Despite being associated with periorbital aging, tear trough deformities are increasingly observed in younger individuals, giving them a fatigued expression. 7Surgical interventions may present challenges in this population including overcorrection and prolonged recovery periods, making filler injections an appealing alternative. 8aluronic acid (HA) is currently the preferred filler for tear troughs, with other options like calcium hydroxyapatite and collagen also available. 9Due to its low elasticity and enzymatic solubility, HA is recommended as the injectable filler of choice for the periorbital region. 8However, superficial HA, commonly used for tear trough injections, may result in the Tyndall phenomenon, a bluish discoloration under the skin in the injected area of the lower lid. 10Although corrections using hyaluronidase or laser treatments are possible, 11 the outcomes are not promising and may impose additional financial burdens on patients.To avoid the Tyndall phenomenon while ensuring effective outcomes, we the use of a combination of HA and collagen for tear trough filling.

| Patients
All patients who participated in the trial signed an informed consent form.This study adhered to the Declaration of Helsinki, and ethics committee approval was obtained.The inclusion criteria were as Group A received HA (Restylane; Galderma, Uppsala, Sweden), Group B received a combination of HA (Restylane) and collagen (Sunmax; Sunmax Biotechnology Co., Ltd), and Group C received collagen (Sunmax).All patients underwent evaluation using the Barton grading system prior to injection.Furthermore, tear trough scores before and after injections were evaluated using the Tear Trough Rating Scale (TTRS), 13 and Investigator Global Aesthetic Improvement Scale (GAIS) 14 scores were assessed following injection.The physicians conducting the evaluations were blinded to the patient subgroups.

| Treatment
The same senior physician performed all procedures.Patients were instructed to clean their faces and apply 5% lidocaine cream for surface anesthesia 30 min prior to the injection.The patients were positioned in a semisitting position throughout the injection procedures.Injections were performed in the periosteal and superficial layers of the orbicularis oculi muscle.HA was injected into the infraorbital medial, central, and lateral regions at three points for patients in Group A. (Figure 1) The periosteum layer was injected using a 27-gauge sharp needle, followed by targeting the superficial layer of the orbicularis oculi muscles, with a dosage of 0.05-0.1 mL injected at each site.In Group C, collagen was injected using a 25gauge cannulas at the intersection of the tear trough line and the lateral canthus straight line.The injection targets the periosteum layer and superficial layer of the orbicularis oculi muscle, utilizing F I G U R E 1 Illustrating the three sites for HA injections to correct the tear trough, located in the medial, medial, and lateral infraorbital regions.fan-shaped technique for both layers, with a unilateral injection volume of 0.5-1 mL, administered from deep to shallow and medial to lateral.(Figure 2) In Group B, both collagen and HA were injected using the same method as in Groups A and C, but into different layers.Specifically, collagen was injected into the superficial layer of the orbicularis oculi, while HA was injected into the periosteum layer.For patients in Group B and C, allergy testing was performed before collagen injection by injecting a small amount of collagen into the palm of the forearm and observing any allergic reactions, such as erythema or itching, following the injection.

| Assessment items
We assessed treatment outcomes based on the following criteria:

| Postinjection complications
Common complications after injection such as edema, bruising, and lumps were recorded.

| Objective assessments
We assessed changes in patients' TTRS scores before injection and at 1 and 3 months postinjection.

| Statistics of the Tyndall phenomenon
The number of cases of the Tyndall phenomenon occurring within three months after injection were recorded.

| Photo evaluation
We used standardized photographs of the patients before and after the injections to evaluate the efficacy of tear trough injections and gather relevant data.During photography, the patient stood upright, looking straight ahead, and maintained a horizontal frankfurter plane.The patient's posture, expression, and camera focus were set uniformly.To maintain the original proportions, all photographs underwent uniform modifications using Adobe Photoshop CC (Adobe Systems Inc., San Jose, CA).We also asked other doctors to evaluate the randomized photos to assess the injection effect in an unbiased manner.

| RE SULTS
A total of 60 patients (seven men and 53 women) were equally divided among the three study groups.The mean age of the patients was 28.57years.Prior to injection, there were no significant differences in age, Barton Tear Trough grades, or TTRS scores among the three groups of patients.Detailed baseline information is presented in Table 1.Postinjection complications were recorded for all three patient groups; some patients experienced two or more complications.(Table 2) The Tyndall phenomenon was observed in six patients in Group A, while none in Group B or Group C experienced it.These differences were considered statistically significant (Table 3).The TTRS scores of the tree groups did not show significant differences at 1 month postinjection compared to the preinjection TTRS scores (p = 0.858).However, there was a significant difference in in the TTRS score 3 months after injection (p = 0.015) (Table 4).
When the differences were specifically analyzed, the results showed significant differences between Groups C and A (p = 0.004) and between Groups C and B (p = 0.045), but not between Groups A and B (p = 0.303) (Table 5).The GAIS scores were not significantly different among the three groups of patients at 1 month postinjection but showed significant differences at 3 months postinjection (p = 0.02) (Table 6).Specifically, analysis revealed no significant difference between Groups A and B (p = 0.748), but a significant difference between Groups A and C (p = 0.024) and Groups B and C (p = 0.010) (Table 7).

| Case 1
A 31-year-old female patient presented with Barton grade I tear trough deformity, with a pretreatment TTRS score of 3. HA injectable fillers were applied to the periosteal and superficial layers of the orbicularis oculi muscle, with a final HA injection volume of 2.5 mL.
Unfortunately, 3 days after the injection, the patient experienced Tyndall's phenomenon, characterized by a blue discoloration in the injected area of the lower lid.The Tyndall effect was promptly resolved using hyaluronidase to dissolve HA in the superficial layer of the orbicularis oculi muscle.After 15 days, collagen was applied for secondary filling, and favorable outcomes were achieved (Figure 3).

| Case 2
The 24-year-old female patient presented with Barton grade II, and had a pretreatment TTRS score of 3. A combined approach using TA B L E 1 Patient demographics before injection.

Group
Variation, Median (P25, P75) *The mean difference is significant at the 0.05 level.
HA and collagen was employed for filling.Collagen was injected at the level of the superficial orbicularis oculi muscle, and HA injected at the level of the periosteum.HA was injected at a final volume of 1.5 mL, and collagen was injected at a final volume of 0.5 mL.This combination of injections yielded excellent therapeutic results and achieved desirable cosmetic morphology (Figure 4).This study has some limitations.First, although this was the first study to assess the efficacy of HA and collagen in filling the tear trough, the sample size was small, and the study involved only one center, limiting the generalizability of the findings.Second, the follow-up period in this study was short.If it had been prolonged to 6 months or even a year, it is probable that the distinction between

| DISCUSS ION
follows: (1) patients aged 20-40 years; (2) Grade I or II on the Barton grading system 12 with no notable lower lid fat protrusion; (3) normal lower lid support structures with no evidence of lid-globe separation; and (4) complete understanding of the research and voluntary signing of an informed consent form.The exclusion criteria were as follows: (1) patients with periorbital or cutaneous diseases; (2) lower eyelids that underwent additional procedures, such as lasers, face ultrasound, radiofrequency, botulinum toxin, or filler injections within the last 12 months; (3) allergy to collagen or HA fillings; and (4) patients who discontinued the study midway.Between June 2022 to January 2023, 60 patients with tear trough deformities were enrolled in the study and randomly assigned into three groups of 20 individuals, each receiving a different injectable filler treatment.

2. 3
.1 | Baseline data This included the patient's age, sex, and Barton grading system grade for the tear trough deformity.Grade 0: Absence of medial and lateral lines demarcating the arcus marginalis or the orbital rim, a smooth youthful contour without a transition zone at the orbitcheek junction; Grade I: mild, subtle presence of medial line or shadow: smooth lateral transition of the lid-cheek junction; Grade II: Moderate prominence of a visible demarcation of the lid-cheek junction, extending from medial to lateral; Grade III: severe demarcation of the orbit-cheek junction, with an obvious step between the orbit and the cheek; and pretreatment Tear Trough Rating Score (TTRS): which measure the distance from the anterior lacrimal crest to the depth of the trough; each millimeter of depth is given 1 point.Hyperpigmentation: no hyperpigmentation, 1 point; mild pigmentation, +2 points; moderate pigmentation, +3 points; and intense or deep hyperpigmentation, +4 points.Prolapse of the nasal fat pads or pockets was rated as mild (1 point), moderate (2 points), or severe (3 points).Lower eyelid skin rhytidosis was rated from 1 to 4 (mild, moderate, advanced, and severe, respectively).
Data analysis was performed using IBM SPSS Statistics (IBM SPSS 26.0,IBM Corp., Armonk, NY, USA).We used the Kruskal-Wallis F I G U R E 2 Illustrating a fan-shaped injection technique for collagen injection to correct tear trough deformities, with the entry site located at the intersection of the tear trough line and the lateral canthus straight line.H test and Nemenyi test to analyze changes in the TTRS and GAIS scores in the three groups.The results were presented as medians and interquartile ranges.Intergroup variability in the Tyndall phenomenon was analyzed using the chi-square test.Statistical significance was set at p < 0.05.
Tear trough filler injection presents one of the most challenging procedures in facial rejuvenation owing to the multifaceted nature of periorbital aging, involving changes in bone, ligament, muscle, fat, and skin.Consequently, each patient's tear trough exhibits different clinical characteristics, necessitating a thorough and methodical evaluation by the doctor, as well as specific treatment approach.Furthermore, to achieve a balanced facial aesthetic, doctors must understand the structural changes induced by aging and have excellent aesthetic perception abilities.We believe that the effects of injectable facial fillers can be categorized into direct and indirect effects.The primary function is to improve the volume deficit in the area by filling pertinent depressions, and the additional function is the myomodulatory effect.Myomodulation operates based on the tonic reflex theory.By injecting fillers deeper into the muscle, the distance between the muscle's starting and ending points can be extended to support the muscle and strengthen its contraction ability when facial muscle contraction becomes weaker.Conversely, if there is excessive muscle contraction, the filler can be injected into the superficial layers of the muscle or close to the point of contraction initiation, thereby inhibiting muscle contraction.15Collagen has long been used as a dermal filler.Collagen was the second dermal filler, after fat, to gain Food and Drug Administration (FDA) approval in the 1980s.Bovine was the first source of collagen; later, porcine and human collagen were created and also received FDA marketing approval.16Despite the emergence of numerous alternative fillers in recent years, collagen has demonstrated great therapeutic benefits, both when used alone and in combination.Collagen has been used for more than three decades and is one of the safest and most effective fillers in the cosmetics market.Currently, HA is the main filler used in research for tear trough injections.However, the occurrence of the Tyndall phenomenon after HA tear trough injections demands attention.Tear trough injections are commonly administered in the superficial and periosteal layers of the orbicularis oculi muscle.Fillers at the superficial orbicularis oculi muscle are primarily used to treat fine wrinkles on the lower lid and, to a lesser extent, volume deficiencies.By contrast, periosteal-level are primarily used to fill depressions and address volume deficits.Numerous scholars believe that the Tyndall phenomenon is the result of HA injections performed at shallow depths.17The injection of HA at a superficial level to treat fine wrinkles on the lower lid may lead to a bluish discoloration under the skin when HA.Even when injected only at the periosteal layer, a small number of patients may still experience Tyndall phenomenon.We hypothesize that the action of the orbicularis oculi muscle over time, small amounts of HA may flow into the superficial layers, causing discoloration of the skin of the lower lid.Although the Tyndall phenomenon can be treated with hyaluronidase or laser irradiation, these options increase the financial burden on patients.Therefore, to repair tear trough abnormalities, we recommend a combination of superficial collagen injections, deep HA injections, and collagen injections.Our findings demonstrated no significant difference in the changes in TTRS and GAIS scores among the three groups at 1 month postinjection.However, one limitation of collagen injections into the tear trough is their shorter retention time, which often does not exceed 3 months.Consequently, compared to the other groups, patients in Group C had significantly different TTRS and GAIS scores at 3 months postinjection.While the comparable scores of patients in Group B did not differ substantially from those in Group A, this may be related to the longer retention of HA in deeper layers, leading to greater volume replacement.

TA B L E 5 *
Multiple comparisons of TTRS variation.TA B L E 6Global Aesthetic Improvement Scores.The mean difference is significant at the 0.05 level.