The Immunologic Spectrum of Biostimulators and Its Clinical Importance

Abstract

Background:

Biostimulators have become important tools in aesthetic medicine to address age-related volume loss and tissue changes. They stimulate the body’s natural processes to produce collagen and other components that contribute to a youthful appearance. Understanding the immunologic mechanisms underlying these processes is crucial for achieving optimal clinical outcomes. We thus sought to review the immunologic mechanisms underlying the action of biostimulators and their implications in clinical practice in aesthetic medicine.

Methods:

A comprehensive literature review was conducted to examine the diverse immunologic mechanisms triggered by commonly used biostimulators, including poly-l-lactic acid, polycaprolactone, and calcium hydroxylapatite, with a particular focus on their physicochemical properties and clinical effects.

Results:

Biostimulators elicit variable wound-healing immune responses based on their physicochemical properties. Injecting a biomaterial recognized immunologically as nonself will follow a foreign body pathway, producing outcomes that can vary from those of an immunologically familiar biomaterial. The extent of tissue regeneration is influenced primarily by the injected biomaterial’s physicochemical properties, and particle size and shape. Other factors (eg, injection technique and contamination) can also influence outcomes. Biostimulator choice depends on specific clinical goals and patient characteristics. All of these factors require consideration when formulating treatment strategies for tissue regeneration.

Conclusions:

Biostimulators elicit a spectrum of immunologic responses dependent on their physicochemical properties, ultimately producing clinical outcomes tending toward replacement or regeneration of native tissue. Understanding their immunologic mechanisms allows for optimal selection and use to achieve desired outcomes. Further research is needed to elucidate the complex immune responses to different biostimulators.

Takeaways

Question: Each biostimulator’s distinct physicochemical properties result in different immune responses. Polymer-based stimulators trigger foreign body reactions, whereas ceramic-based fillers promote regeneration. Clinicians must develop a good understanding of these immunologic mechanisms to optimize biostimulatory filler use.

Findings: The immune response involves complex interactions between macrophages, giant cells, and other immune cells. Immune responses to fillers depend on biomaterial composition, particle characteristics, and the local microenvironment. Injection technique, contamination, and patient factors influence biostimulator outcomes.

Meaning: Different biostimulators lead to distinct outcomes: tissue replacement or regeneration. Ceramic-based stimulators, such as calcium hydroxylapatite, may offer advantages in restoring aging tissue structure and function.

INTRODUCTION

Aesthetic medicine addresses the visible signs of aging such as volume loss and loss of tissue integrity. Hyaluronic acid (HA) has been a popular product for replenishing lost volume and injecting into dermal wrinkles. However, despite low complication rates, HA can cause delayed inflammatory responses¹ and facial overfill syndrome,² and has prompted a shift toward injectable biostimulators that induce the autologous synthesis of tissue components such as collagen to replace lost volume, referred to as replacement biostimulation. Regenerative aesthetic procedures restore age-related tissue loss with more youthful structure and function. This is achieved with biostimulators that restore the dynamic and architectural ecosystem of the extracellular matrix (ECM). It benefits clinicians to understand the underlying biological processes that influence clinical outcomes for appropriate product selection.

BIOSTIMULATORS: DEFINITIONS, CLASSIFICATIONS, AND FUNCTIONS

Biostimulation is a broad term describing the process of stimulating enhanced biological processes in cells and tissues, promoting healing, regeneration, and overall physiological function over multiple medical indications, including soft tissue and bone loss due to trauma or surgery. It is also involved in improving wound healing in ulcers caused by chronic disease, such as diabetes or postradiation injury. Biostimulation encompasses devices such as low-level laser therapy, which uses specific wavelengths of light to stimulate cellular activity,³ and electric microcurrents, which have shown promise in healing chronic ulcers and surgical wounds.⁴ Biostimulants also include biological and synthetic materials that serve as scaffolds for tissue regeneration. These biostimulant scaffolds are widely used in medical applications, including periodontal treatments, cardiac valves, and joint replacements. In these applications, their presence enhances healing processes by providing a conducive environment for cell attachment, migration, and proliferation.^5,6 Advanced techniques include the addition of stem cells to biostimulatory scaffolds in a synergistic approach combining regenerative therapies⁶ and biocues from platelet-rich plasma to direct immune responses in wound healing. Additionally, exosomes, which may provide an off-the-shelf source of biocues, are of great interest but currently remain approved only for topical use by regulatory authorities due to unresolved issues.^7–9

BIOSTIMULATORS: REPLACEMENT TO REGENERATIVE SPECTRUM OF ACTIVITY

Injectable biostimulatory biomaterials direct reparative physiological pathways toward either end of a wound healing spectrum. At the extreme replacement end, innate collagen fills areas of volume loss, causing thick, fibrotic scar tissue.¹⁰ At the other end of the wound healing spectrum, regeneration occurs when all native structural elements of the tissue ECM are produced in correct and native ratios, architectures, and proportions^10–12 to support a healthy cellular population. True regeneration is exemplified by salamanders¹³ that regrow amputated limbs and by intrauterine scarless wound healing.¹⁴ The outcomes of biostimulation lie in the spectrum tilting more toward replacement or regeneration. Biostimulation outcomes can be influenced by the biomaterial, patient’s genetics and health status, injecting technique, and contaminants.^10,15 To gain a better understanding of the biological process of biostimulation and how the selection of biomaterials can influence clinical outcomes, we performed a comprehensive literature review of poly-L-lactic acid (PLLA), polycaprolactone (PCL), and calcium hydroxylapatite (CaHA) biostimulators. The schematic in Figure 1 illustrates this concept as well as others described in the subsequent sections. As shown, an immediate response occurs after injection of polymer and ceramic biostimulants, leading to neutrophil recruitment and protein adsorption onto the biomaterial surface. During the subsequent immune response, polymer surfaces trigger a foreign body response (FBR), whereas ceramic CaHA surfaces activate regenerative macrophages and promote ECM renewal.

Fig. 1.

Schematic of the spectrum of tissue regeneration or replacement associated with biostimulators. DAMPs, damage-associated molecular patterns; PAMPS, pathogen-associated molecular patterns; VEGF, vascular endothelial growth factor.

CLINICAL BENEFITS OF REPLACEMENT AND REGENERATIVE BIOSTIMULATORS

Aging skin results from the interplay of many factors, including loss of tissue integrity and soft tissue–supporting ligamentous structures, fat atrophy, sarcopenia, and bone loss and remodeling. Replacement of volume loss with HA is common but is associated with short longevity, hydrophilicity causing swelling, and potential inflammatory reactions. Consequently, patients now request the replacement of the volume loss with their own collagen. This autologous volumization can be achieved by injecting replacement-orientated¹⁶ biostimulators.^17,18 Enhancement or restoration of tissue integrity, strength, support, pliability, elasticity, and vascularity requires the selection of a biostimulator that promotes both structural and functional regeneration.

BIOSTIMULATORY BIOMATERIALS USED IN AESTHETICS

Injectable biomaterials possess different physicochemical properties, degradation profiles and pathways, and biological interactions. Ceramic biomaterials, including calcium hydroxyapatite, are well tolerated in vivo and elicit minimal immune responses.¹⁹ They are crystals composed of inorganic elements such as oxygen, calcium, and phosphorous. Immune responses to ceramics occur due to tissue trauma from implantation and involve macrophages; however, inflammation resolves without chronic progression.²⁰ Bioactive ions²¹ released during the degradation of ceramics modulate the reparative immune microenvironment.

Polymers are large molecules of repeating small units bonded into long chains. PLLA and PCL can be manufactured with differently shaped and sized particles. However, they are immunologically distinct because they elicit an FBR.²² They follow foreign body immune pathways that produce predominantly collagenous deposition around the implanted material. This replacement process with collagen is leveraged in aesthetics, where the replacement of volume loss is achieved via the injection of polymer biomaterials.

IMMUNOLOGIC REPAIR PATHWAYS AND THE INFLUENCE OF INJECTED BIOMATERIALS

The complex immunologic repair pathways that occur in wound healing and physiological responses to injected biomaterials²³ are finely tuned continual processes that comprise 4 transitional phases. In the first phase, thrombus or platelet plugs form immediately in response to local trauma and foreign biomaterials, with different biomaterials influencing plug composition.²⁴ During the inflammatory phase, to defend immediately against bacteria, immune cells such as neutrophils migrate to and infiltrate the thrombus.²⁵ The thrombus releases cytokines and chemokines that attract circulating monocytes,²⁶ which differentiate into macrophages that infiltrate the area, phagocytose debris, combat infection, and direct subsequent immunologic repair pathways. This point is critical to the subsequent immunologic pathways, as it is determined by the particle size and shape, physicochemical composition, and the potential presence of bacterial contaminants and cellular damage through excessive trauma. Macrophage responses to the thrombus vary with foreign materials and microbes but influence the continued cytokine release and attract immune cells, including mast and T-helper cells.²⁷ The proliferation phase involves angiogenesis and recruitment of activated fibroblasts to produce the structural components of primitive granulation tissue.²⁸ Depending on where the biostimulator lies on the spectrum, tissue remodeling²⁹ subsequently produces the final outcome toward replacement (via haphazard deposition of collagen) or regeneration (via restoration and remodeling toward the original tissue form and function, with minimal scarring).

THE THROMBUS PLUG ON A BIOMATERIAL

Proteins including fibrinogen, albumin, immunoglobulins, fibronectin, and complement proteins adsorb onto a biomaterial surface. The pattern and type of protein adsorption depend on the particle’s physicochemical characteristics,³⁰ including surface hydrophilicity/hydrophobicity, chemistry, charge,³⁰ nanotopography,³¹ mechanical stiffness,³² and the Vroman effect³⁰ (the dynamic repositioning of the proteins on the biomaterial surface) at the biomaterial interface. Immune cells have receptors on their surfaces that recognize these proteins and affect their response.³³ Different biomaterials induce different proteomic coatings and thus influence the cellular responses that determine the outcomes of immunologic repair pathways.

MACROPHAGES AND MULTINUCLEATED GIANT CELLS

During thrombus (a provisional matrix) formation, circulating monocytes are recruited to the injury site, respond to local environments, and differentiate into macrophage subsets with different functions and roles in wound healing and tissue regeneration.²⁷ This differentiation is not terminal and allows macrophages to exhibit phenotypic plasticity. Pro-inflammatory M1 macrophages express cytokines (interleukin [IL]-6, IL-1, tumor necrosis factor-alpha) that recruit other inflammatory cells, including T cells and mast cells. Toll-like receptors help recognize biomaterials as self or nonself and adsorbed proteins triggering macrophage activities and immune response cascades. An in vitro study³⁴ showed that M1 and M2 macrophage inflammatory activity was consistently absent with CaHA, whereas inflammatory cytokine expression increased with PLLA, suggesting that CaHA neither induced M1 polarization and inflammatory responses in macrophages nor activated toll-like receptors. Although a recent study³⁵ suggested that both PLLA and undiluted CaHA upregulated gene pathways for the regeneration of the ECM, this is not supported in other studies.

Following thrombus resolution,²⁶ recruited inflammatory cells and intercellular messages switch inflammatory M1 macrophages to regulatory and repair M2 phenotypes.^36,37 IL-4 from mast cells and T-helper 2 (Th2) cells and IL-13 from Th2 cells promote polarization to profibrotic M2a macrophages. M2c regulatory macrophages secrete IL-10 to dampen inflammation and modulate responses.³⁶ Excessive M2a activity causes excessive collagen production.³⁷ Balanced macrophage responses and phenotypes drive outcomes toward either replacement or regeneration.

At the biomaterial interface, macrophages can fuse into multinucleated giant cells (MNGCs).³⁸ Macrophage phenotypes and MNGC subsets at the biomaterial interface behave and interact differently based on the physicochemical and morphological properties of the biomaterial. M1–M2a macrophages that recognize foreign components may fuse into a subset of MNGCs known as foreign body giant cells (FBGCs). FBGCs are associated with the FBR that encapsulates the biomaterial in fibrous tissue,²⁷ and they persist while the biomaterial is present. FBGC formation, although poorly understood, likely results from cellular responses to direct biomaterial physicochemical characteristics, such as size and shape, stimuli such as IL-4 and IL-13, and a recognized pattern of adsorbed proteins on the biomaterial surface. Other immune cells influencing MNGC polarization include profibrotic Th2 and T-helper 1 cells involved in antibacterial defense. However, fibrosis is directly related to the number of macrophages and FBGCs associated with the biomaterial.³⁹ The different properties of ceramic and polymer biomaterials influence the differentiation of inflammatory cells into different phenotypes of MNGCs. The variability of multinucleated cells associated with biomaterials⁴⁰ means that their presence does not inherently indicate a fibrotic FBR.

Osteoclasts are another subtype of multinucleated cells characterized by a ruffled border,⁴¹ which is not seen in other MNGC subsets. They can derive from monocytes, macrophages, or dendritic cells and be involved in immune disorders. An ATPase proton pump in the ruffled border membrane releases hydrogen ions into the lacunae, leading to the dissolution of bone minerals. They are also strong stimulators of angiogenesis.⁴¹ Different subsets of integrins (transmembrane proteins by which cells adhere to the ECM) are involved in macrophage attachment to different biomaterials and in MNGC fusion at the biomaterial surface.⁴¹ The large, fused cells with ruffled borders observed at the CaHA microsphere interface are most likely osteoclast-like multinucleated giant cells (OLMNGCs) or a related subset of MNGCs that are phenotypically different from FBGCs and are M2c-associated.³⁸ FBGCs observed to be associated with a polymer such as PLLA are part of a fibrotic replacement pathway with collagen. The OLMNGCs observed at the interface of the ceramic CaHA-carboxymethylcellulose (CMC) microspheres, which are immunologically recognized as self, do not cause FBR.^42,43 Rather, they have a different role of extracellular breakdown into its ionic components, tissue regeneration, and angiogenesis. High surface-to-volume ratio particles have more macrophages and FBGCs than smooth-surface implants, indicating that M1 polarization is less likely with smooth, round particles.⁴⁴

At the biomaterial interface, M1 macrophages and M1–M2a MNGCs release acidic agents, reactive oxygen species, and enzymes⁴⁵ that break down the biomaterial. With prosthetic devices, this causes device failure; however, with injected, nonpermanent polymeric biostimulatory compounds, this breakdown into smaller particles may be beneficial, allowing the MNGCs to phagocytose degraded biomaterial particles so that the replacement pathway stimulus is not permanent.⁴⁶

PROLIFERATION AND REMODELING

Stimulus (biomaterial) persistence prevents the resolution of repair pathways. During the proliferative phase, fibroblasts are recruited, and angiogenesis occurs through macrophage and inflammatory cell signaling within the provisional matrix.²¹ The different proliferative and remodeling responses are predetermined by the initial immune inflammatory phase in the presence of biomaterials. Granulation tissues⁴⁷ form and are remodeled in FBR replacement pathways toward mature fibrous tissue, in regeneration pathways with appropriate structural components and architectures, or somewhere in between. In the presence of a nonself biomaterial such as a polymer or a biomaterial that has large jagged particles, a proinflammatory M1 MNGC (FBGC) predominates.⁴⁸ These cells ultimately switch to a reparative, anti-inflammatory phenotype M2a MNGC and express transforming growth factor beta to recruit and activate fibroblasts to produce a collagen-dominated fibrotic matrix.^33,49 Conversely, if the biomaterial is recognized as self, such as CaHA, and has a smooth round surface, a different regenerative pathway occurs involving regulatory T ⁵⁰ cells, stem cells, and local growth factors.⁵¹ Therefore, different biomaterials can elicit distinct immune responses and outcomes.

Poly-l-lactic Acid

PLLA is an aliphatic polyester thermoplastic polymer⁵² that induces collagen production via FBR⁵³ and was initially used for volume replacement in treating HIV-associated lipodystrophy. The Sculptra PLLA comprises heterogenous, 2–200 μm PLLA flakes.⁵⁴ The combination of polymeric nonself chemistry and a high surface-to-volume ratio promotes macrophage M1 polarization.⁴⁸ Furthermore, particles less than 10 µm in size are phagocytosed by M1 macrophages and dendritic cells and presented to the lymph nodes, triggering the adaptive immune system to recruit further immune cells and prolong the inflammatory phase.⁵⁵ FBGCs form at the biomaterial interface, whereas less-organized collagen type III encapsulates the particles.⁵⁶ Current evidence suggests that as PLLA gradually metabolizes into lactic acid monomers, newly synthesized collagen replaces the lost volume,⁵⁷ providing structural support to the skin. The newly formed collagen is remodeled to a mature collagen network, resulting in long-lasting soft tissue support and volume replacement.⁵⁸

Polycaprolactone

PCL, a polyester polymer currently used as a replacement biostimulator, also initiates FBRs at its interface. It first degrades into polymers small enough to be engulfed by macrophages and then further degrades into 6-hydroxycaproic acid units to be metabolized in the liver.⁵⁹ Poly-ɛ-caprolactone (Ellansé; Sinclair Pharmaceuticals, United Kingdom) injectable PCL microspheres degrade slowly over several years,⁶⁰ allowing a prolonged FBR immune pathway until the stimulus is removed.

CaHA

CaHA is the only injectable ceramic biostimulator and interacts with the immune system differently than polymer biostimulators. Although the precise mechanism of CaHA-CMC (Radiesse; Merz Aesthetics, Raleigh, NC) remains to be elucidated, it does not rely on inflammatory pathways, avoids FBRs, and facilitates tissue regeneration. Collagen production increased in cultured fibroblasts directly exposed to CaHA-CMC microspheres, indicating a direct mechanotransduction effect.^15,61 Fibroblasts recognize CaHA microspheres and attach to them through integrins.⁶² The immune response directs the recruited and activated fibroblasts into regenerative pathways.⁶³ With no FBR to CaHA,⁶⁴ M2c macrophages and regulatory T cells can drive a pro-regenerative immune response without an excessive M2a fibrotic response.⁶³ This process encourages activated fibroblasts to produce ECM components in the correct native ratios and to remodel them into the correct native 3-dimensional tissue architectures,^65–67 thus restoring functions such as pliability and dermal thickness.⁶⁷ Large cells likely to be OLMNGC abutting the microspheres degrade the microspheres into microgranules, which disperse throughout the ECM and subsequently into calcium and phosphate ions.⁴³ The release of calcium likely influences macrophage polarization and promotes regenerative processes.⁶⁸ In the bone, biphasic calcium phosphate releases calcium ions that induce long-term M2 macrophage polarization via the Wnt/β-catenin signaling pathway.⁶⁹ This occurs through the activation of calcium-sensing receptors in macrophages, which subsequently promotes mesenchymal stem cell differentiation.⁶⁹ Adipose-derived stem cells also interact directly with calcium phosphate bioceramics,⁷⁰ further enhancing their regenerative mechanisms.⁷¹ CaHA-CMC has been demonstrated to promote angiogenesis that results in a healthy glow to the skin.^66,72 The osteoclast subset of MNGCs stimulates angiogenesis, and it is possible that OLMNGC play a similar role.⁴¹

OTHER FACTORS THAT INFLUENCE IMMUNOLOGIC RESPONSES TO BIOSTIMULATORS

Macrophage phenotype switching is influenced by crosstalk with other cells and the local microenvironment. This has mostly been demonstrated in in vitro studies; however, many in vivo factors are also involved. Injectors must be aware of these factors when selecting and predicting biostimulator injection outcomes. A patient’s genetic profile can affect cytokine responses and immune reactions, emphasizing the influence of genetic variation on immune pathways. Thus, individual responses to biostimulator injections vary. Aging alters cellular functions, local microenvironments, and immune responses. Immunosenescence, dysregulation of macrophage function, and delayed resolution of immune responses have been demonstrated to be associated with aging and can prevent the desired clinical outcome from biomaterial injections.⁷³ Immunosuppression through disease or pharmaceutical interventions significantly influences the immune response to biomaterials. Tissue trauma associated with biomaterial injections produces damage-associated molecular patterns that are recognized by pattern recognition receptors on macrophages and dendritic cells. Excessive trauma and associated damage-associated molecular patterns stimulate an inflammatory response that can skew immune responses toward M1 polarization and encourage macrophage fusion at the material interface. This may explain why FBGCs were observed in ischial pressure areas treated with CaHA.⁷⁴ Contamination of the biomaterial can introduce bacterial lipopolysaccharides and lipoproteins, which are potent stimulators of macrophages toward M1 polarization and fusion into FBGC at the biomaterial interface,⁷⁵ leading to a profibrotic FBR. Thus, an aseptic technique is essential when injecting biostimulators. Despite nonsterile techniques being often reported as contributing to late complications of filler injections,⁷⁶ the use of optimum aseptic techniques when injecting biostimulators is still infrequent, but is relevant if the regenerative properties of an injected biomaterial such as CaHA are desired. Biostimulator rheology can also affect outcomes; stiffness affects cell fate and function, and can influence macrophage polarization.^77,78 Biomaterial dilution also affects outcomes.¹² Diluted CaHA-CMC (1:1) improved biostimulatory properties¹⁵ by increasing the accessibility of CaHA-CMC microspheres to cells, although this may also be related to the lower G’ and reduced M1 polarization.⁷⁸ Dilution of PLLA reduces inflammation.⁷⁹ Large bolus injections are associated with granuloma formation, indicating an exaggerated inflammatory response.⁸⁰

CLINICAL IMPLICATIONS OF REGENERATIVE AND REPLACEMENT BIOSTIMULATORS

Replacement biostimulators use collagen to restore volume lost during aging, but the collagen lacks the normal ECM architecture. In contrast, regenerative biostimulators restore tissue function and structure by producing collagen, elastin, and other structural elements in the correct ratios and architectures, thus supporting a cellular ecosystem. Dysregulated inflammation can cause fibrosis, a pathological feature of many diseases.⁴⁹ Excess collagen deposition stiffens the ECM, disrupts cell signaling, and causes cellular dysfunction. Biostimulators can be injected intradermally or into the superficial and deep fat pads of the face to restore volume. Facial fat compartments provide volume, structural support, thermal regulation, metabolism, protection, and tissue repair. Fat cells also cross-communicate with other facial tissues.⁸¹ Thus, the functional implications of using a biostimulator that is primarily a “collagen stimulator” must be considered, for performing predominantly volume restoration. Conversely, aesthetic regenerative biostimulators restore appearance and volume while retaining fat pad pliability and ECM architecture. In the skin, collagen replacement can smoothen wrinkles but produces a stiff dermis, whereas regenerative orientated biostimulators such as CaHA-CMC produce a thicker, more elastic dermis with good vascularity, improved function, pliability, and hydration.⁶⁷ However, as discussed, factors such as bacterial contamination, concurrent pathologies, or excess trauma can shift a regenerative outcome toward a fibrotic response. Understanding how the choice of biomaterial influences outcomes toward replacement (more fibrotic) or regeneration (renewal of normal tissue) has implications for future surgical dissection procedures. Biostimulators with more regenerative potential, such as CaHA-CMC at 1:1 and 1:2 dilutions, are less likely to disrupt targeted surgical planes.

CONCLUSIONS

Biostimulators are increasingly being used to address volume deficits by stimulating native collagen production. Understanding the immunologic pathways elicited by a particular biomaterial is essential. Immune cells identify the injected biomaterial as either self or nonself, prompting reparative immunologic responses toward replacement or regeneration. Nonself-recognized biostimulators, such as PLLA, induce chronic inflammatory FBRs and collagen production to compensate for age-related volume loss. In contrast, self-recognized biostimulators trigger repair mechanisms that restore tissues with identical elements, thereby reinstating the structure, function, and volume lost through aging. Injectable biostimulator biomaterials in aesthetic medicine lie on a spectrum between replacement and regeneration. However, factors such as bacterial contamination, tissue trauma, or concurrent pathology can shift immunologic repair pathways toward inflammatory replacement and fibrotic outcomes.

DISCLOSURE

Corduff and Goldie are paid clinical consultants and lecturers for Merz Aesthetics. Funding for manuscript editing and preparation was provided by Merz Asia Pacific Pte Ltd to Dr. Shawna Tan, Medical Writers Asia.