Biostimulating fillers and induction of inflammatory pathways: A preclinical investigation of macrophage response to calcium hydroxylapatite and poly-L lactic acid.

INTRODUCTION: Initial macrophage response to biostimulatory substances is key in determining the subsequent behavior of fibroblasts and the organization of newly synthesized collagen. Though histological studies suggest that calcium hydroxylapatite (CaHA) filler initiates a regenerative healing response with collagen and elastin deposition similar to natural, healthy tissue rather than an inflammatory response with fibrosis, the relative activity of macrophages stimulated by CaHA, as well as how this activity compares to that induced by other biostimulatory fillers, has not been explored. The aim of the study is to characterize the in vitro macrophage response to two biostimulory fillers, CaHA and PLLA (poly-L lactic acid), and to evaluate their inflammatory potential. METHODS: Primary human macrophages were incubated with two dilutions (1:50 and 1:100) of commercially available CaHA or PLLA. After 24 h incubation, an inflammation array was used to screen for the expression of 40 cytokines, released by macrophages. ELISA was used to confirm array results. RESULTS: Four cytokines were significantly upregulated in M1 macrophages incubated with PLLA compared to both unstimulated controls and CaHA: CCL1 (p < 0.001), TNFRII (p < 0.01), MIP-1α (p < 0.05), and IL-8 (p < 0.001). In M2 macrophages, MIP-1α (p < 0.01) and MIP-1ß (p < 0.01) were significantly upregulated by PLLA compared to CaHA and unstimulated controls. CONCLUSION: Together, these findings indicate that the CaHA mode of action is a non-inflammatory response while PLLA initiates expression of several cytokines known to play a role in inflammation. Our study supports the concept that these two "biostimulatory" fillers follow distinct pathways and should be considered individually with regard to mechanism of action.

Evaluation of Paste-Type Micronized Acellular Dermal Matrix for Soft Tissue Augmentation: Volumetric and Histological Assessment in a Mouse Model.

BACKGROUND: A biological injectable material, paste-type micronized acellular dermal matrix (ADM), has been proven effective in wound healing by filling defects through tissue replacement. This study aimed to compare the efficacy of paste-type micronized ADM on soft tissue augmentation with that of the conventional fillers in animal experiments. METHODS: Two distinct paste-type micronized ADMs, which were mixed with distilled water (mADM) and gelatin (mADM+GEL), respectively, were compared with conventional fillers, hyaluronic acid (HA) and polymethyl methacrylate (COL+PMMA). Thus, four different types of fillers were each injected into the dorsum of nude mice to compare the volume retention and biocompatibility. During the 8-week experimental period, ultrasound and computed tomography (CT) images were obtained for volumetric analysis. Histological evaluation was performed using hematoxylin and eosin and CD 31 staining. RESULTS: According to the CT images at week 8, the mADM and mADM+GEL showed a higher volume persistence rate of 113.54% and 51.12%, compared with 85.09% and 17.65% for HA and COL+PMMA, respectively. The 2-week interval ultrasound images revealed that the mADM showed a volume increase in width rather than in height, and an increase in height for HA did not vary much. Histological analysis showed marked fibrous invasion and neovascularization with the mADM and mADM+GEL compared to that of the conventional fillers. CONCLUSIONS: Paste-type micronized ADM showed soft tissue augmentation with similar effectiveness to that of conventional fillers. Therefore, paste-type micronized ADM has potential as an alternative material for a soft tissue filler in tissue replacement. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Evaluation of Adverse Effects of Resorbable Hyaluronic Acid Fillers: Determination of Macrophage Responses.

Resorbable tissue fillers for aesthetic purposes can induce severe complications including product migration, late swelling, and inflammatory reactions. The relation between product characteristics and adverse effects is not well understood. We hypothesized that the degree of cross-linking hyaluronic acid (HA) fillers was associated with the occurrence of adverse effects. Five experimental HA preparations similar to HA fillers were synthesized with an increasing degree of cross-linking. Furthermore, a series of commercial fillers (Perfectha®) was obtained that differ in degradation time based on the size of their particulate HA components. Cytotoxic responses and cytokine production by human THP-1-derived macrophages exposed to extracts of the evaluated resorbable HA fillers were absent to minimal. Gene expression analysis of the HA-exposed macrophages revealed the responses related to cell cycle control and immune reactivity. Our results could not confirm the hypothesis that the level of cross-linking in our experimental HA fillers or the particulate size of commercial HA fillers is related to the induced biological responses. However, the evaluation of cytokine induction and gene expression in macrophages after biomaterial exposure presents promising opportunities for the development of methods to identify cellular processes that may be predictive for biomaterial-induced responses in patients.

Stem Cell-Derived Extracellular Vesicle-Bearing Dermal Filler Ameliorates the Dermis Microenvironment by Supporting CD301b-Expressing Macrophages.

Hyaluronic acid-based hydrogels (Hyal-Gels) have the potential to reduce wrinkles by physically volumizing the skin. However, they have limited ability to stimulate collagen generation, thus warranting repeated treatments to maintain their volumizing effect. In this study, stem cell-derived extracellular vesicle (EV)-bearing Hyal-Gels (EVHyal-Gels) were prepared as a potential dermal filler, ameliorating the dermis microenvironment. No significant differences were observed in rheological properties and injection force between Hyal-Gels and EVHyal-Gels. When locally administered to mouse skin, Hyal-Gels significantly extended the biological half-life of EVs from 1.37 d to 3.75 d. In the dermis region, EVHyal-Gels induced the overexpression of CD301b on macrophages, resulting in enhanced proliferation of fibroblasts. It was found that miRNAs, such as let-7b-5p and miR-24-3p, were significantly involved in the change of macrophages toward the CD301bhi phenotype. The area of the collagen layer in EVHyal-Gel-treated dermis was 2.4-fold higher than that in Hyal-Gel-treated dermis 4 weeks after a single treatment, and the collagen generated by EVHyal-Gels was maintained for 24 weeks in the dermis. Overall, EVHyal-Gels have the potential as an antiaging dermal filler for reprogramming the dermis microenvironment.

Sodium Alginate as a Potential Therapeutic Filler: An In Vivo Study in Rats.

Filler injection demand is increasing worldwide, but no ideal filler with safety and longevity currently exists. Sodium alginate (SA) is the sodium salt of alginic acid, which is a polymeric polysaccharide obtained by linear polymerization of two types of uronic acid, d-mannuronic acid (M) and l-guluronic acid (G). This study aimed to evaluate the therapeutic value of SA. Nine SA types with different M/G ratios and viscosities were tested and compared with a commercially available sodium hyaluronate (SH) filler. Three injection modes (onto the periosteum, intradermally, or subcutaneously) were used in six rats for each substance, and the animals were sacrificed at 4 or 24 weeks. Changes in the diameter and volume were measured macroscopically and by computed tomography, and histopathological evaluations were performed. SA with a low M/G ratio generally maintained skin uplift. The bulge gradually decreased over time but slightly increased at 4 weeks in some samples. No capsule formation was observed around SA. However, granulomatous reactions, including macrophage recruitment, were observed 4 weeks after SA implantation, although fewer macrophages and granulomatous reactions were observed at 24 weeks. The long-term volumizing effects and degree of granulomatous reactions differed depending on the M/G ratio and viscosity. By contrast, SH showed capsule formation but with minimal granulomatous reactions. The beneficial and adverse effects of SA as a filler differed according to the viscosity or M/G ratio, suggesting a better long-term volumizing effect than SH with relatively low immunogenicity.

Practical Guidelines for Hyaluronic Acid Soft-Tissue Filler Use in Facial Rejuvenation.

BACKGROUND: Hyaluronic acid (HA) fillers are the most commonly used fillers for soft-tissue augmentation. The face is a dynamic structure. Facial rejuvenation by filler products depends on mechanical forces on the region of the face. The successful use of injectable HA fillers requires an understanding of the options available. OBJECTIVE: The purpose of this study is to measure the rheological properties of HA fillers and to clarify how to select these fillers considering their rheological properties. MATERIALS AND METHODS: Rheological characterization was performed on 41 fillers. Physical parameters directly linked to product performance were measured. RESULTS: The properties of the HA fillers varied. These findings provide a basis for selection guideline regarding rheological properties in facial rejuvenation. CONCLUSION: The authors' report is the largest study to determine the rheological properties of HA fillers to date. Understanding the fillers' properties can help physicians select the appropriate fillers for more predictable and sustainable results.

Hyaluronic acid-cross-linked filler stimulates collagen type 1 and elastic fiber synthesis in skin through the TGF-ß/Smad signaling pathway in a nude mouse model.

Compared to pure hyaluronic acid filler, cross-linked hyaluronic acid (HAc) exhibits superior biocompatibility and longevity as a dermal filler. We previously developed composite HAc-hydroxyapatite (HAp) fillers. Herein, we systematically compared the protein-level increase and gene expression between HAc-micro-HAp and HAc-nano-HAp in mice and determined the mechanisms underlying the biological responses to HAc and HAp. Five-week-old female BALB/c-nude mice were classified into five groups: normal skin, Radiesse, Restylane, HAc-nano-HAp, and HAc-micro-HAp. Fillers (200 µl) were injected to evenly fill the back of mice. Skin biopsies were performed to investigate collagen and elastic fiber synthesis after filler injections. Western blot analysis, real-time polymerase chain reaction analysis, and immunohistochemistry were performed to investigate protein and gene expression changes. Organ (liver, lung, spleen, and kidney) toxicity of HAc-nano-HAp was determined by hematoxylin and eosin staining after 12 weeks. Protein and gene expression analyses indicated that, compared with pure fillers, HAc-nano-HAp and HAc-micro-HAp hydrogels preferentially promoted collagen and elastic fiber formation through the TGF-ß pathway. The composite fillers also exhibited no evidence of organ toxicity. HAc-HAp filler might play an important role in collagen and elastic fiber regeneration. HAc filler stimulates collagen type 1 and elastic fiber synthesis through the TGF-ß/Smad pathway. The role of HAc-HAp composite fillers in photoaging in animal models and their effects on skin, including elasticity and tensile strength, should be investigated.

Comparative Evaluation of the Biodegradability and Wrinkle Reduction Efficacy of Human-Derived Collagen Filler and Hyaluronic Acid Filler.

BACKGROUND: The development of fillers for wrinkle prevention is growing to meet rising demands to reduce the aging of skin. OBJECTIVE: In this experiment, we confirmed the effects of human collagen and hyaluronic acid filler biodegradation for wrinkle reduction using a photo-aging mouse model. MATERIALS AND METHODS: A total of 10 hairless mice (SKH1-Hrhr) were randomly divided into two groups and injected with hyaluronic acid and human-derived collagen filler. At 0, 2, 4, 8, and 12 weeks, PRIMOSlite®, folliscope, and MRI were used to evaluate the biodegradability of the fillers after the injections. We also studied the photo-aging mouse model for skin roughness and histological evaluation and confirmed that the filler injection had excellent anti-wrinkle effects. RESULTS: Human-derived collagen fillers had excellent biodegradability compared to that of hyaluronic acid fillers. The skin surface roughness in the photo-aging mouse models was significantly reduced after injections of human-derived collagen filler. CONCLUSION: Our results showed that the human-derived collagen filler had excellent biodegradability and effectively reduced wrinkle formation in a photo-aging mouse model. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Nonsurgical Rhinoplasty: Nasal Grid Analysis and Nasal Injecting Protocol.

BACKGROUND: Nonsurgical rhinoplasty using injectable fillers improved skin quality and texture, cost-effectiveness, and quick recovery. The aim of this study was to provide a valid and customized protocol for injecting the nose with the aid of a nasal grid. METHODS: From January of 2016 to October of 2017, 150 consecutive patients entered the trial. The mean patient age was 36 years (range, 16 to 60 years). The patients were divided into two arms: primary nasal defects (no previous surgery, n = 109) and secondary nasal defects (previous surgery, n = 41). The primary defect group included 43 male patients and 66 female patients, and the secondary nasal defect group included 14 male patients and 27 female patients. Within each arm, patients were stratified into three age group tiers: younger than 30 years, 30 to 45 years, and older than 45 years. RESULTS: The grid became the reference for quantity and sequence of injection records. The overall complication rate was 1.82 percent, and a visual analogue scale ranging from 1 to 10 was implemented (with 10 being the most accurate correction). Of the patients evaluated, 98.350 percent scored 8 to 10, 0.825 percent scored 6 to 8, and 0.825 percent scored below 6. CONCLUSIONS: Fillers can be either alternatives or complementary to plastic surgery. Nonsurgical rhinoplasty, with its avoidance of general anesthesia, splints, swelling, and bruising, represents a distinct advantage, allowing the patient to return to work as quickly as the same or the next day. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Biochemistry, Physiology, and Tissue Interactions of Contemporary Biodegradable Injectable Dermal Fillers.

BACKGROUND: Injectable dermal fillers are becoming increasingly popular for soft tissue augmentation and rejuvenation. Most contemporary biodegradable products are derived from hyaluronic acid, calcium hydroxylapatite, or poly-L-lactic acid. Achievement of desired cosmetic outcomes is largely dependent on selection of the optimal injectable product based on the chemical composition, the physiologic interactions with surrounding tissue, product longevity, and a thorough understanding of potential adverse reactions. OBJECTIVE: To review and describe the biochemistry, physiology, and tissue interactions of the most commonly used contemporary biodegradable dermal fillers. METHODS: A thorough review of the literature was performed with additional review of pertinent clinical cases and corresponding histopathology. RESULTS: This article provides a comprehensive review of the biochemistry, physiology, and potential tissue interactions of the most commonly used biodegradable dermal fillers. The underlying biochemical properties of each product and how they contribute to specific physiologic and adverse tissue reactions is described. CONCLUSION: Understanding of the innate differences in the physical properties, and physiologic responses to soft tissue fillers allows clinicians to achieve desired aesthetic outcomes with fewer adverse events.

Evaluation of the In Vivo Kinetics and Biostimulatory Effects of Subcutaneously Injected Hyaluronic Acid Filler.

BACKGROUND: Because subcutaneously injected hyaluronic acid filler is absorbed over 6 months to 1 year after the treatment of facial wrinkles, frequent retreatment may be required. However, persistent long-term effects are often clinically observed when hyaluronic acid filler is injected as a bolus for facial augmentation. Therefore, the authors investigated, over time, the changes in volume and histologic features of subcutaneous bolus injections of hyaluronic acid. METHODS: Hyaluronic acid filler was subcutaneously injected as a bolus into the dorsum of 6-week-old rats. At several time points (immediately after injection and 4, 8, 16, 32, and 64 weeks thereafter), magnetic resonance imaging was introduced to observe morphologic changes and to measure volume. Histologic examination of sectioned tissues was also performed. RESULTS: The average volume increased for up to 4 weeks after injection and then gradually decreased, with 74.8 percent of the injected volume remaining after 64 weeks, with no statistical difference compared to the initial volume. Histologic analysis revealed that lattice structures were created by fibroblasts and collagen fibers, and blood vessels and adipocytes were also generated in the filler. CONCLUSIONS: Although subcutaneous bolus injections of hyaluronic acid filler exhibited flattening, the total volume was maintained even after 64 weeks. Histologically, hyaluronic acid filler acted as a scaffold for autogenous tissue replacement by means of fibroblast migration and proliferation, collagen induction, and angiogenesis, followed by proliferation of adipocytes. This study demonstrates that the total volume is maintained long-term by replacing part of the injected hyaluronic acid filler with autologous tissues.

CALCIUM HYDROXYLAPATITE MICROSPHERES - BIOCOMPATIBILITY AND CLINICAL EFFECTS.

Soft tissue fillers are used in aesthetic medicine for volumizing and for contouring. Calcium hydroxylapatite (CaHA) is a fully biodegradable biostimulatory filler. The study presents results of in vitro biocompatibility and cytotoxicity investigations performed on CaHA and illustrates its clinical effects. We used a human cell culture model for in vitro studies with HaCaT keratinocytes and human dermal fibroblasts, known to be a sensitive cell type for biocompatibility and cytotoxicity. Cells were exposed to different concentrations of CaHA (Radiesse®). Cell proliferation was calculated by luminometric adenosine triphosphate (ATP) measurement using the ATPLiteTM-M Assay. Possible cytotoxic effects were detected by the calorimetric Cytotoxicity Detection Kit. Clinical data were obtained from our own treatment files. CaHA did neither inhibit cell proliferation nor cause cytotoxicity. Clinical data suggest an excellent tolerability and long-term efficacy superior to hyaluronic acid-based fillers. CaHA is a versatile and well-tolerated biodegradable and biostimulatory filler.

Fluorodeoxyglucose activity associated with a cosmetic poly-L-lactide filler: a potential confounder on positron emission tomography and computed tomography.

Injectable cosmetic fillers are increasingly popular, but are not often considered as a cause of abnormal findings on imaging. We present a case of poly-L-lactic acid (PLLA) filler associated with 18-fluorodeoxyglucose (FDG) uptake, which had the potential to interfere with staging of a squamous cell carcinoma of the lateral tongue. We characterise the FDG-positron emission tomography/computed tomography (PET/CT) properties of a PLLA dermal filler, Sculptra® (Aventis), and highlight its potentially confounding appearance in the staging scans of oncological patients, particularly those with oral tumours.

Tissue Augmentation in Wound Healing: the Role of Endothelial and Epithelial Cells.

INTRODUCTION: Wounds and their complications present a frequent cause of morbidity and mortality in everyday clinical practice. In order to reduce the wound burden, much effort has been directed into the physiology of healing and new therapeutic approaches. AIM: This paper provides an overview from the literature about the role of endothelial and epithelial cells in tissue filler employment for wound healing. MATERIAL AND METHODS: The scientific literature was reviewed through PubMed, Medline and Science Direct. The articles were chosen in correlation with the study objective and their scientific relevance. RESULTS: Successful wound healing depends on many diverse processes, cell types and molecular mediators. The definitive aim of wound healing is a properly healed wound. Tissue fillers are becoming an important alternative in wound management, although augmentation of soft tissue can present a demanding problem due to the difficulties in tissue survival. In order to prevent its failure, an optimal vascular network needs to form from wound edges into the filler. CONCLUSIONS: Because of the importance of chemotaxis and angiogenesis in various physiological and pathological processes, both events present an extensive area of intense research. Additionally, epithelial cells are needed to cover the wound defect and sealing the wound environment from outer world.

Reversible Alopecia with Localized Scalp Necrosis After Accidental Embolization of the Parietal Artery with Hyaluronic Acid.

Hyaluronic acid (HA) filler injection is widely used for soft-tissue augmentation. Complications associated with HA filling are not uncommon; however, HA-induced alopecia is a rarely reported complication that could result in severe secondary psychological trauma. The etiology, clinical traits, treatment strategies, outcomes, and possible reversibility of HA-induced alopecia have not been characterized. Here, we report a case in which bilateral temple injections of 6.5 mL of HA led to persistent pain over the left scalp for several days. Although the pain was relieved at day 9 after 600 U of hyaluronidase were injected in the left temple, the patient developed localized alopecia at the left temporoparietal region with central skin necrosis at day 15. After topical applications of recombinant bovine basic fibroblast growth factor gel and 2% minoxidil spay, the necrotic skin wound was healed at day 42. Hair regrowth and normal hair density were restored at day 74. Analyses of Doppler ultrasound examinations and histopathology of the skin biopsy suggested that mild ischemia of the left temporoparietal region led to reversible alopecia, while the permanent hair loss in the left parietal area was associated with severe skin ischemia. Therefore, the key to treatment would be to focus on the effective correction of severe ischemia-induced skin necrosis to prevent permanent hair loss. Level of Evidence V This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Periorbital and Intraorbital Studies of the Terminal Branches of the Ophthalmic Artery for Periorbital and Glabellar Filler Placements.

BACKGROUND: Filler injections for sunken upper eyelid correction and glabellar augmentation at the orbitoglabellar region need to be performed correctly. Precise knowledge of the emerging sites of all terminal branches of the ophthalmic artery is essential for these procedures to be conducted safely. METHODS: The terminal branches of the ophthalmic artery were studied in both periorbital and intraorbital dissections. The aim of this study was to verify the critical positions of the emerging sites at the orbital septum that may act as potential retrograde channels for filler emboli. RESULTS: In the 40 eyes examined, the branches of the ophthalmic artery were found to emerge from four different sites. Two substantial emerging sites were situated on both sides of the trochlea of the superior oblique muscle. These sites were located at the superior part of the medial orbital rim (SMOR) and are alternatively named as the epitrochlear and the subtrochlear emerging sites. The other two sites can be regarded as accessory emerging sites due to the comparably smaller artery. Dissection of the intraorbital region revealed small periosteal branches of the infraorbital artery which coursed anteriorly on the orbital floor to form anastomoses with the lacrimal artery. In other areas of the orbital floor, no branches extended from the infraorbital artery. In front of the lacrimal gland, very minute branches descended and coursed along both margins of the superior tarsus but did not course outside the lateral orbital rim. CONCLUSION: A danger zone was located at the SMOR, where the ophthalmic branches emerge to form anastomotic channels. Compression at the trochlea guarantees safe injection of filler, reducing the risk of complication. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Clinical Application of Earlobe Augmentation with Hyaluronic Acid Filler in the Chinese Population.

BACKGROUND: Larger earlobes, which are a symbol of "richness" in traditional Chinese culture, are favored by Chinese patients. The objective of this paper is to investigate the application of earlobe augmentation with hyaluronic acid (HA) filler injection and its clinical effects in the Chinese population. METHODS: A total of 19 patients (38 ears) who received earlobe augmentation with HA filler injections between March 2013 and March 2015 were included. The clinical effects, duration, and complications of these cases were investigated. RESULTS: All patients who received earlobe HA injections showed immediate postoperative effects with obvious morphological improvement of their earlobes. The volume of HA filler injected into each ear was 0.3-0.5 ml. The duration of the effect was 6-9 months. Two of the 19 cases (3 ears) demonstrated mild bruising at the injection site, but the bruising completely disappeared within 7 days after the injection. No vascular embolism, infection, nodule, or granuloma complications were observed in the studied group. CONCLUSION: The application of earlobe augmentation with HA filler injection is a safe, effective, simple procedure for earlobe shaping. It has an easy clinical application with good clinical prospects. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .