The histologic reaction and permanence of hyaluronic acid gel, calcium hydroxylapatite microspheres, and extracellular matrix bio gel

The filling materials on the beauty market can be classified into three types: natural biological materials, synthetic polymer materials, and composites containing bioactive substances. However, comparative experimental data is lacking to compare their biological responses and permanence.

and promote a series of self-repair processes in the body to achieve antiaging effects.Recently, the last stage combines materials with bioactive materials to produce composites such as extracellular matrix (ECM) 6 and tissue engineering scaffolds 7 containing seed cells.
In addition to the first, the remaining three types of materials are widely used in clinical practice.
The ideal soft tissue filling materials should be soft, have good biocompatibility, safety, controllable body reaction, maintain a stable and lasting effect, and can be completely degraded.After injection, the fillers will stimulate the inflammatory response.The inflammatory cells and inflammatory factors such as TNFα, IL-1, and IL-6 are recruited, and the phagocytosis of macrophages is triggered. 8A controllable inflammatory response is beneficial.It provides protection for our bodies.If it gets out of control, it can lead to the formation of granulomas.In order to understand the biological response to different fillers, we chose HA, CaHA, and ECM as the representative material for these three types of materials, and we performed a study in mice evaluating the histologic changes seen at 1, 4, 8, 16, and 24 weeks after injection.

| Soft tissue fillers and animals
Hyaluronic acid gel (Bloomage Biotechnology Corporation Limited).

CaHA microspheres (MOYANG Biotechnology) which consist of
CaHA microspheres suspended in a gel made of water, glycerin, and sodium carboxymethylcellulose.ECM bio gel (Beijing Dikang Pharmaceutical Investment Management Co., Ltd) was extracted from the greater omentum of the porcine.Six-week-old female C57BL/6N mice (WeitongLihua Laboratory Animal Technology Co.,

Ltd).
The mice were divided into four groups: saline control group, HA group, CaHA microspheres group, and ECM group.To reduce the difference caused by different injection sites, the four groups of fillers were injected into the subcutaneous back of 32 mice in a clockwise direction according to the volume of 0.1 mL.At 1, 4, 8, and 16 weeks after the injection of fillers, the test site was removed completely for further experiments, including the injected fillers, the peripheral tissue capsule, and the whole skin.

| Volume retention rate
The vernier caliper was used to measure the fillers' length (2a), width (2b), and height (c).The formula for hemiellipse was used to calculate the volume V and the retention rate 0δ of the mass. 9,10The higher the δ, the higher the volume retention rate, the lower the absorption rate, and the longer the effect.Record its dynamic changes.

| Immunohistochemistry
The specimens were fixed in 4% formaldehyde and embedded in paraffin for routine histology.Thin sections were prepared and stained with hematoxylin-eosin (for inflammatory cells), Masson (for collagen), and CD31 (for neovascularization) staining according to standard immunohistochemistry protocols.The sections were scanned by a panoramic scanner (3DHISTECH, Hungary) and processed with CaseViewer2.4(3DHISTECH, Hungary).The dermal thickness of the skin around the injection was measured continuously at the same intervals at week 16.
A total of five points were measured in each section.
Two pathologists blindly evaluated the local foreign body response of all slides according to the Duranti histologic score, 11 as follows: 0: no visible reaction, 1: slight inflammatory reaction with a few inflammatory cells, 2: clear inflammatory reaction with one or two giant cells, 3: fibrous tissue with inflammatory cells, lymphocytes, and giant cells, and 4: granuloma with encapsulated implant and clear foreign body reaction.
Images were captured using fluorescence microscopy (Nikon).

| ELISA
The following commercial immunoassay kits were used to detect protein levels in samples: TNFα ELISA Kit, IL-1β ELISA Kit, IL-6 ELISA Kit (Thermo, USA), VEGF ELISA Kit (MULTISCIENCES, China), and

Collagen I and Collagen III ELISA Kit (Jianglai Biological Technology
Co., Ltd., China).The samples were homogenized mechanically under an ice bath and centrifuged at 3000 rpm for 10 min.The supernatant was taken for further determination following the instructions.Optical density (OD) was measured at 450 nm using a microplate reader (Biotek Instruments Co., Ltd).A standard curve was made according to the concentration and OD value of the standards, and then calculated the concentrations of all samples based on the standard curve.

| Statistical analysis
The data were expressed as the mean ± SD and analyzed using either Student's t-test or ANOVA with a Tukey's post hoc test by GraphPad Prism software.A p < 0.05 was considered statistically significant.

| Appearance evaluation and volume retention rate
All three fillers could form a subcutaneous mound after being injected into the back of mice.No complications such as infection, ulcer, erythema, or severe edema occurred in all animals.In addition, hair grew back normally over the subcutaneous mass.Among the three groups, the mass of the ECM group had the softest texture, followed by the HA group, and the CaHA group was the hardest.
However, due to the ECM bio gel was not encapsulated after injection, it is difficult to maintain the original shape, and it is hard to be measured.We only compared the volume retention rate between the HA gel and the CaHA microspheres.
The results showed that the volume retention rates of the HA gel groups were significantly higher than those of the CaHA microspheres group at 1 and 4 weeks (p < 0.01).At 8 weeks, there was no statistical difference in the volume retention rates between the two groups (p = 0.44).Until 16 weeks, the HA gel group had a lower value than the CaHA microspheres group, which was 20.24% and 26.03% (p < 0.05).The volume of the HA gel groups maintained a relatively stable decline from the beginning to the end.For the CaHA microspheres group, the volume decreased more rapidly than the other, but it significantly slowed over time.The rapid degradation and absorption of the sodium carboxymethylcellulose in microspheres at an early stage may have contributed to the problem (Figure 1).

| HE staining
The histologic pictures of all fillers were similar at 1 week.They only showed a slight inflammatory response with few lymphocytes and granulocytes around the injection (Duranti grade 1).HA and CaHA were well encapsulated by collagen fibers, while ECM can hardly be encapsulated.For ECM and HA groups, the number of inflammatory cells was negligible at all times, suggesting that ECM and HA induce a minimal immune response (Duranti grade 1).Still, for the CaHA group, a large number of macrophages, multinucleated giant cells, and other inflammatory cells around the microspheres showed signs of clear inflammatory reaction and active microspheres phagocytosis at 4 and 8 weeks (Duranti grade 3).However, at a later stage, the dwindling number of inflammatory cells represented the foreign body response was still under control (Figure 2; Table 1).

| ELISA of TNFα , IL-1β, and IL-6
The ELISA analysis of TNFα, IL-1β, and IL-6 showed that the results were basically consistent with the histologic analysis.At the early stage, the inflammatory response was slight, and only the CaHA groups showed increased expression of all three inflammatory factors.At 4 weeks, all fillers can promote the expression of proinflammatory mediators, but the protein concentration decreases with time.The expression of all three factors in the CaHA group was significantly higher than that in other groups during the first 8 weeks.At 16 weeks, there was no noticeable difference among the four groups, meaning the inflammatory responses induced by these fillers were controllable.All fillers have good biocompatibility and would not cause excessive or long-term reactions (Figure 3).

| CD31 staining
The CD31 staining results at 4 weeks showed the newly formed blood vessels in the gap between the CaHA particles.At 16 weeks, the neovascularization was more evident than at 4 weeks.The ingrowth of vessels and connective tissue could be seen in the periphery of the CaHA microspheres.This phenomenon may be due to the special porous structure of the microspheres, which provides a suitable scaffold for neovascularization.At the 8 weeks, a small amount of neovascularization could also be seen in HA gel.And for the ECM group, the formation of new blood vessels is almost invisible around the injected fillers (Figure 4).

F I G U R E 1
The volume retention rate of HA and CaHA at 1, 4, 8, and 16 weeks.The results represent the mean ± SD.CaHA, calcium hydroxylapatite; HA, hyaluronic acid.
In contrast, the results of the HA and the CaHA groups were significantly increased compared with the control group.At week 16, collagen expression in the CaHA group was higher than in the other two groups.
The results of thickness measurement at week 16 showed that all three fillers could thicken the local dermis.The mean value of the ECM group (149.49± 24.63 μm) is slightly higher than the control group (130.13 ± 20.93 μm) but less than the HA group (159.27 ± 26.81 μm) and the CaHA group (159.64 ± 19.04 μm) (Figure 5).

| DISCUSS ION
HA, one of the most common fillers, is universally present in all species and creates volume by binding water.The inflammatory response of HA after injection was mild, and no adverse reactions such as granuloma and infection were observed.Its degradation rate is stable and good volume can be maintained.Also, HA can slightly stimulate neovascularization.Vessel growth is integral to tissue repair, as vessels support local cells through nutrients and oxygen, which contribute to tissue repair and regeneration. 12lcium hydroxyapatite is the main component of bone.It has been used as bone cement for a long time, which proves its safety.
Because of its physical nature, it is hard to remain soft in the body.
It is more suitable for filling the deeper layers of tissue.Studies have shown that the foreign body response caused by the injection of a cell-free substance can polarize macrophages toward the M2 type and release TGFβ, which stimulates fibroblasts to produce collagen fibers. 13CaHA can induce collagen and vessel formation around the microspheres as a long-term stimulatory filler.However, due to the characteristics of its gel carrier, its early volume retention decreased too fast to confirm the initial injection volume in clinical application.
The ECM 14 has good biocompatibility and hardly induces body reactions, even when used in allogeneic transplantation. 15,16It has its natural structure and important bioactive components such as collagen, laminin, and growth factors, which can provide a good support structure. 17In this study, the ELISA analysis showed a temporary increase in levels of inflammatory factors after injection.And in the clinic, some early allergic reactions happen to a minority of patients injected with ECM, such as injection-site swelling, itching, and erythema. 18,19It is possibly due to the residual cellular components or detergents.At week 16, dermal thickness in the ECM group was slightly higher than that in the control group, which may be caused by the growth factors contained in the ECM.

| CON CLUS ION
In this study, three widely used materials on the market were comprehensively compared in terms of their permanence and histologic reaction in vivo.All the tested materials are illustrated to be biocompatible and histologically safe.Each of the three types of fillers has its advantages and disadvantages.ECM is pliable, with a high degree of similarity to natural soft tissue and minimal body reaction.
The immunohistochemistry and immunofluorescence results indicated that collagen deposition in the periphery of the CaHA microspheres occurred at 4 weeks.For the HA group, collagen formation occurred at 8 weeks.The expression of collagen increased with time.At 16 weeks after injection, many collagen-encapsulated microspheres could be observed in the center and periphery area of the injected substance in the CaHA group.Collagen III is the main component of new collagen fibers in the fillers.ELISA analysis showed no significant difference in the expression levels of two kinds of collagen in the tissues around the injected fillers in the ECM group and the control group at different periods.
However, it is difficult to maintain the original volume and position after injection.HA has excellent biocompatibility but lower volume retention than CaHA in the long term.CaHA is a longer lasting filler to induce new collagen formation.It tends to generate palpable nodules when injected into the superficial layer of the skin.Therefore, F I G U R E 5 (A) Masson staining and immunofluorescence of Collagen I and Collagen III.Bar = 100 μm.(B) ELISA result of Collagen I and Collagen III.(C) Dermal thickness of four groups at 16 weeks.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.CaHA, calcium hydroxylapatite; Col, Collagen; ECM, extracellular matrix; HA, hyaluronic acid.