Dilutional rheology of Radiesse: Implications for regeneration and vascular safety

Calcium hydroxylapatite‐carboxymethylcellulose (CaHA‐CMC) injectables have emerged as dual‐purpose fillers with bioregenerative and direct filling capabilities.


| INTRODUC TI ON
6][7] The biostimulatory effect of CaHA-CMC gels is attributed to the mechanical interactions between CaHA microspheres and dermal fibroblasts, which are further potentiated through aqueous dilution of the product prior to injection. 1,210][11] In practice, CaHA-CMC is considered dilute at a 1:1 ratio and hyperdilute above a 1:1 dilution ratio.However, the extent to which aqueous dilution of these gels impacts their rheological properties and potential behavior within tissues has not been described.In this study, we investigate the effect of increasing dilution volume on the rheological properties of CaHA-CMC dermal filler and its CMC carrier gel, with a focus on the product's storage modulus (G′), loss modulus (G″), complex viscosity (η*), loss factor (tan δ), cohesivity, and extrusion force.

| ME THODS
This study employed CaHA-CMC (Merz Aesthetics, Raleigh, NC, USA), a dermal filler composed of synthetic CaHA microspheres suspended in a gel carrier of sterile water, glycerin, and sodium carboxymethylcellulose (CMC), manufactured according to the valid Bill of Materials by Merz North America, Wisconsin.In addition, a CaHA microsphere-free CMC gel was formulated for experimental assessment of the carrier gel component.The CMC gel (Lot No. CMC02063023, Merz R&D, Frankfurt, GE) was prepared by the Filler and Medical Device Development department (Merz, Frankfurt, GE) composed of CMC (Lot No. R3003267, CP Kelko, Aanekoski, Finland), glycerin (Lot No. K099B, Riedel de Haan, Seelze, GE), and demineralized water.Glycerin was first pre-mixed with CMC using a spatula.Subsequently, water was added and mixed overnight using a Turbula shaker.The gel was loaded into a 1.5 mL CaHA-CMC syringe in aluminum pouches, sealed, sterilized at 121°C and stored until use.The composition of the formulated CMC gel strictly conformed with the CMC gel of CaHA-CMC identically.

| Preparation of CaHA-CMC and CMC gel dilutions
Samples of diluted CaHA-CMC and CMC gel carrier were prepared using the amount of CaHA-CMC or CMC carrier gel and saline as described in Table 1.For all dilutions used except undiluted, 1.5 mL of each gel was transferred into a suitable mixing syringe (5 mL or 10 mL Luer Lock syringe, Becton Dickinson and Company, Franklin Lakes, USA) via a Luer-to-Luer connector (Baxter RAPIDFILL Connector, Baxter Healthcare, Deerfield, USA).Using the smallest graded syringes adequate for each dilution volume, the described volume of 0.9% saline was drawn into the second mixing syringes.The following volumes of saline were added to produce undiluted, 1:0.25, 1:0.5, 1:1, 1:2, and 1:3 dilution samples: 0, 0.38, 0.75, 1.5, 3, and 4.5 mL, respectively.It is worth noting that the FDA-approved 0.26 mL dilution (1:0.17) was not investigated in this study as it breaks the uniform dilution steps.All visible air bubbles were removed from the syringe with gravitational precipitation, and the contents of the two mixing syringes (one with 1.5 mL of CaHA-CMC or CMC gel carrier, the other with 0.9% saline) were homogenized by depressing the plungers with at least 20 mixing strokes (1 stroke = 2 pump moves).After mixing, each solution was visually inspected for homogeneity and immediately tested.

CaHA-CMC and diluted CMC gels
Diluted and non-diluted CaHA-CMC and CMC gels were rheometrically assayed for multiple viscoelastic attributes, including storage modulus (G′), loss modulus (G″), complex viscosity (η*), loss factor (tan(δ)), cohesivity, and extrusion force.These rheological properties impact the physical behavior and clinical performance of filler gels; their meaning and practical implications are outlined in Table 2 and visualized in Figure 1.

| Extrusion force measurements
Undiluted CaHA-CMC was measured directly out of the injection syringe after attachment to a 27 G ¾" Terumo thin wall needle.
Diluted CaHA-CMC was transferred back into the original CaHA-CMC syringe immediately after mixing and attached to a 27 G ¾" needle.The syringes of each dilution were placed in a rig of the texture analyzer (TA.XTplus Texture Analyzer, Surrey, UK), and the test was started with the following parameters (Figure S1A).
Under the compression setting, the piston head progressed at 12.6 mm/min with an automated trigger force of 0.

| Cohesivity measurements
The drop weight test was performed using a TA instruments texture analyzer.A 18G 1 1/2" Terumo needle with a plane orifice at a speed of 7.8 mm/min was used in the test.The drop weight test was performed five times (five times sampling of 10 droplets) for each dilution and for undiluted CaHA-CMC.The amount of 10 drops was measured, and the average of one drop in milligram was calculated.The amount of gel of one drop corelated positively with the cohesivity of the sample.Initially, this test was developed for hyaluronic acid (HA) fillers, which have neglectable difference in density. 12,13However, this is not the case for the CaHA-CMC dilutions due to the presence of CaHA microspheres.The density of the product was considered in the cohesivity assessment, and the results of the drop weight test were calculated in μl/drop instead of the standard mg/drop.A summary of measured densities can be found in Figure S2, and the experimental setup can be found in Figure S3.
TA B L E 2 Rheological measurements, symbols, descriptions, and physico-clinical correlations.

| Statistical analysis
Where appropriate, all data are presented as mean ± standard de- While η* is a more clinically relevant measure of viscous behavior, G″ values are also reported in Table 3.In practice, the viscosity of CaHA-CMC becomes near negligible at a 1:1 dilution.For example, the G′ values of 1:1 dilute CaHA-CMC are nearly half that of activated autologous platelet rich plasma (PRP). 15In addition, consistent with these rheological changes, the loss factor, tan(δ), of CaHA-CMC significantly increased with increasing dilution (Figure 2C).A summary of all rheological values for different dilutions of CaHA-CMC at frequencies ranging from 0.1 to 10 Hz can be found in Figures S4-S9.
The cohesivity of diluted CaHA-CMC progressively decreased with increasing dilution, mirroring the density of the mixture.

| Properties of CMC gel carrier
The rheological properties of undiluted CMC gel carrier, previously undescribed, are summarized in Table 4 and   with mechanically biostimulatory effects.8][19][20] Within this regenerative realm, dilute mixtures of CaHA-CMC have spearheaded this emerging application of CaHA-CMC gels in aesthetic medicine, challenging the conventional uses of this product. 10,21,22CaHA-CMC thus exists on a resiliencyregeneration continuum that makes it unique as both a filler and regenerative biostimulator.
CaHA-CMC, in its undiluted, diluted or hyperdiluted form, is adeptly suited for soft tissue regeneration, promoting the activation of endogenous fibroblasts. 1,2,23The mechanical interaction between fibroblasts and CaHA microspheres is quintessential for de novo tissue formation.However, noting that regeneration is optimized with dilution, it is thus necessary that an adequate amount of space between neighboring microspheres exist for optimal regeneration, with both in vitro and clinical histological analysis supporting this notion. 1,2To this extent, dilutions of 1:1 through 1:3 appear to elicit optimal regenerative effects. 24 In this study, aqueous dilution of CaHA-CMC had a profound impact on the rheology of the gel, with a 1:1 dilution imparting a 98% reduction in G′.Therefore, beyond the 1:1 ratio, dilutions are unlikely to achieve any direct filling effects, with any observable volumization ensuing from the endogenous regenerative response driven by CaHA microspheres. 1,25Functionally, undiluted or hypodiluted mixtures (under 1:1) retained meaningful rheological properties, capable of achieving patient-desired aesthetic soft tissue filling, contouring, and dynamic wrinkle effacement while simultaneously imparting increased regenerative effects. 2 Additionally, this study revealed that CaHA-CMC has meaningful cohesivity when undiluted, but that even small dilution volumes dramatically decrease cohesivity, facilitating its tissue biointegration and stimulatory effects.In the case of microsphere-containing fillers, low cohesivity enables particles to diffuse optimally throughout the treatment area, as the gel carrier surface tension is easier to overcome.Curiously, at hyperdilutions, the cohesivity sustained a marginal increase, possibly due to a decreasing surfactant effect by the CaHA microspheres.
Relatedly, the extrusion force was significantly altered through the first three dilutions, though not proportionately to the G′, which is likely due to the shear-thinning nature of the CMC gel. 16e rheology of the CMC gel on its own revealed that, without CaHA microspheres, the rheology is remarkably inelastic, nonresilient, and non-cohesive.The CaHA microspheres presumably reinforce the CMC via establishment of a well-dispersed CaHA microsphere lattice.In context, the cohesivity of HA fillers is significantly higher, with most ranging in mg rather than the ug observed with CaHA-CMC. 13e findings in this study suggest that the colloidal nature of CaHA-CMC can be tailored to achieve a customized rheological profile that matches the requirements of target tissues.
A nonlinear regression analysis with a highly fit line allows one to accurately tune the desired rheology based on dilution volume (Figure 5A,B).For example, if the desired G′ for a certain CaHA-CMC injection is 800 Pa, the addition of 0.12 mL of diluent should be added.A summary table of dilution volumes required for sequentially decreasing G′ is given in Table 5. Hyperdilutions of CaHA-CMC, on the other hand, can configure the product to enhance its regenerative role. 24This customizable, dual nature of CaHA-CMC can thus be exploited along a continuum existing between a highly resilient filling profile and a maximally pro-regenerative mode (Figure 6), based on data reported by Casabona et al. 2 Calibration of the product to yield a bi-functional profile is also possible with dilutions between 0.1 and 1 mL, in which a truly regenerative filler coexists with a meaningful G′ (Figure 6B).Notably, dilutions past 1:3 do not increase the total amount of de novo collagen, but rather spreads the same quantity of new collagen over a larger tissue volume, that is, dilutions beyond the optimal 1:1-1:3 stimulate a larger tissue volume to a lesser degree. 1,24At 1:3 dilutions, however, CaHA-CMC has ideal dispersion in situ, does not impart immediate volumization, and is capable of driving significant regeneration.
Dilutional changes in the rheology of CaHA-CMC may also have implications for safety, specifically as it relates to the rare instances of vascular occlusion. 26

| CON CLUS IONS
The rheological characteristics of CaHA-CMC gels are significantly impacted by aqueous dilution of the product.Compared to undiluted product, hyperdilution substantially decreases the product's resiliency and cohesivity, leading to improved product dispersibility and is correlated with an enhancement of its previously reported biostimulatory efficacy.However, the dilutional alterations in the product's rheological profile exist along a continuum between maximal filler resiliency and optimal regenerative effects that may be harnessed clinically to enhance the suitability of the product to a

2. 6 | 2 . 7 |
Correlation with biostimulatory dataTo elucidate the role of dilutional rheomodulation on biostimulation, a previously published data set from Casabona et al which measured the total quantities of collagen 1 and collagen 3 from patients treated with increasing dilutions of CaHA-CMC.2Casabona et al.'s methods for quantification were as follows: tissue specimens from CaHAinjection sites were obtained, fixed in formaldehyde, and processed for light microscopy.These sections were stained with picrosirius red to visualize skin layers and for a densitometric study of collagen.Using a Zeiss Axio 5 microscope, collagen fibers were observed, with different colors indicating collagen types.The Digimizer 4.5 application was used to analyze the images, allowing for a quantitative evaluation of collagen types, and ensuring consistent analysis by a single pathologist.Analysis of the MAUDE database An analysis of the Food and Drug Administration's (FDA) Manufacturer and User Facility Device Experience (MAUDE) database, which is a voluntarily submitted device reports, was F I G U R E 1 Visualization of common rheological properties.(A) Visualization of low and high G′ viscoelastic gels.(B) Visualization of increasing tan delta.(C) Visualization of high and low cohesivity gels following straining forces.scrubbed for reports of vascular occlusions within the date range of 6/1/2004 to 7/11/2023, with the first recorded incidences appearing in 2013.The following codes were considered to fit possible vascular occlusion symptoms: obstruction/occlusion, occlusion, visual impairment, embolism, unspecified vascular problem, necrosis, loss of vision, ischemia, eye injury, vascular system, and optical nerve damage.The generated report was further categorized into cases linked to Radiesse or Radiesse (+).A further screen organized cases by dilution data.Entries that were linked to previous reports were not counted twice and were thus excluded from analysis.The reported occlusions were expressed in absolute values, as relative injection rates are not captured in MAUDE reporting.

4 |
DISCUSS IONThe modern application of dermal fillers in aesthetic medicine has evolved into a nuanced science in which rheological properties, anatomical planes, and biological tissue responses are carefully considered, necessitating a tailored approach.This evolved paradigm has favored the formulation of products that are multifaceted, possessing inherent direct filling capabilities and regenerative properties.CaHA-CMC naturally possesses this combination of attributes, featuring a highly resilient colloid matrix of microparticles imbued F I G U R E 2 Rheological measurements of Radiesse measured at 0.7 Hz. (A) Elastic modulus (B) viscosity, (C) tan(δ), and (D) cohesivity of undiluted, 1:0.25, 1:0.5, 1:1, 1:2, and 1:3 diluted Radiesse.

4
While this dilution-dependent regenerative response has been important to our understanding of its mechanism of action, it has raised questions regarding the effects of dilution on the rheological and tissue filling properties of CaHA-CMC.The findings in the current F I G U R E 3 Extrusion forces of different dilutions of Radiesse.Summary data for CMC gel.F I G U R E 4 Rheological properties of different dilutions of CMC gel without CaHA microspheres measured at 0.7 Hz. (A) Elastic modulus (B) viscosity, and (C) tan(δ) of undiluted, 1:0.25, 1:0.5, 1:1, 1:2, and 1:3 diluted CMC.Comparisons of (D) elastic modulus, (E) viscosity, and (F) tan(δ) between CMC gel and Radiesse at increasing dilutions.study enhance our insight into the physical behavior of this product modification.

2 F I G U R E 6
First, aqueous dilution of a colloid gel reduces the amount of potentially obstructive particles per volume of intravascular injectate relative to undiluted product.This decrease in the embolic burden imposed upon a vascular network may lower the injury potential.Second, the rheological modifications imparted by aqueous dilution decrease the product's cohesivity and resilience, increasing fragmentation, dispersibility, and distal clearance of its CaHA/CMC microparticles (25-45 μm).26,27Finally, the significant decrease in the elastic modulus of hyperdilute CaHA-CMC mixtures lowers the gel's ability to resist shear deformation or impose a compressive load, both of which are likely to play a role in intravascularly occlusive and externally compressive mechanisms (Figure 7A,B).For CaHA-CMC, this concept is supported by our analysis of reports of the FDA MAUDE database (Figure 8), in which only 9 (4.8%) reported cases of possible vascular occlusion involved diluted product, compared to 177 undiluted cases F I G U R E 5 Nonlinear regression curve to approximate the dilution volume required to achieve a desired elastic modulus.(A) Dilutional volumes starting at 0.5 mL and (B) 0 mL.(95.2%).Normalizing for the 2.7 million CaHA-CMC injections reported by the American Society of Plastic Surgeons (ASPS), the overall risk of occlusion with undiluted product, already low at 0.0069%, decreases to 0.00033% with any dilution, and to 0.00018% with hyperdilution.Nonetheless, given the voluntary and unsubstantiated nature of the MAUDE database reporting and intrinsic limitations relating to statistics from only ASPS, these numbers provide only an approximate assessment of the overall relative incidence of these infrequent injuries, which remains extremely low.TA B L E 5 Interpolated dilution volumes required to achieve a range of G′ values.The continuum of volumization and regeneration.(A) The continuum of regeneration and neocollagenesis from undiluted to 1:3 and (B) the continuum of regeneration and neocollagenesis from undiluted to 1:1.Blue line represents the elastic modulus of CaHA-CMC measured in this study, while the red line represents the in vivo collagen measured by Casabona & Pereira. 2

F I G U R E 8
range of applications.This dilutional rheomodulation of CaHA-CMC thus offers a potentially valuable tool increasing product versatility while maximizing safety.AUTH O R CO NTR I B UTI O N S A.M. designed the research study, analyzed the data, and wrote the manuscript.D.S. analyzed the data, wrote, and edited the manuscript.A.C. analyzed the data and assisted in writing and editing the manuscript.R.E.-B.collected the data and assisted in writing and editing the manuscript.N.H. collected the data and assisted in writing the manuscript.All authors have edited the final draft and approved the final manuscript for publication.F I G U R E 7 Models of filler-mediated vascular occlusion.(A) Mechanism of compression and (B) intravascular occlusions.Correlation between dilution and potential vascular complications reported from the MAUDE database between 6/1/2004 and 7/11/2023.