Hyaluronic Acid-Based Hybrid Hydrogel Microspheres with Enhanced Structural Stability and High Injectability.

For hydrogel injection applications, it is important to improve the strength and biostability of the hydrogel as well as its injectability to pass easily through the needle. Making gel microspheres is one approach to achieve these improvements. Granulization of a bulk hydrogel is a common procedure used to form microsized particles; however, the nonuniform size and shape cause an uneven force during injection, damaging the surrounding tissue and causing pain to the patients. In this study, injectable hyaluronic acid (HA)-based hybrid hydrogel microspheres were fabricated using a water-in-oil emulsion process. The injectability was significantly enhanced because of the relatively uniform size and spherical shape of the hydrogel formulates. In addition, the biostability and mechanical strength were also increased owing to the increased cross-linking density compared with that of conventionally fabricated gel microparticles. This tendency was further improved after in situ calcium phosphate precipitation. Our findings demonstrate the great potential of HA-based hydrogel microspheres for various clinical demands requiring injectable biomaterials.

Introduction

Biomaterials have received particular attention for a wide range of therapeutic applications, and many approaches have been introduced to provide multifunctionality by evolving from pure to composite materials.[1,2] Despite these advances, there is a disparity between fabricating hybrid systems and applying them in compliant clinical treatments, such as the practical use of injectable materials.[3−5] Hydrogels have been introduced to meet the demands for injectability because they are easily malleable without cracking and maintain their tissue-like elasticity because of their high hydration properties.[6−8] The injectability of hydrogels allows them to fill three-dimensional spaces in the tissue, thereby broadening their clinical applications from cosmetic products to localized drug delivery and regenerative cell therapies.[4,9−12]

To apply these developments to practical injectable hydrogel applications, granulization of a bulk hydrogel is a common procedure used to transform the gel into microsized particles to meet the demands for injectability.[13,14] The particulated assemblies are usually designed depending on the location or depth of the tissue or the applications.[15,16] In this case, the stiffness and size of the particulated hydrogels critically affect the injection force when they are applied to tissue in vivo.[17] Hyaluronic acid (HA) hydrogels are remarkable biocompatible and biodegradable polymers that are particularly popular for injectable clinical applications.[18−23] HA is a hydrophilic polysaccharide composed of d-glucuronic acid and N-acetyl-d-glucosamine, which is found in the extracellular matrix (ECM) and regulates biological functions for cell proliferation and migration.[24−26] Various chemical reactions have been used to modify HA hydrogel to control their mechanical properties, including their elasticity and degradation resistance.[27] Moreover, by introducing biofunctional molecules or reactive moieties to HA, hydrogels with multifunctionality such as self-healing or shear-thinning properties can be obtained, which provide ease of injectability, leading to their advance in biomedical applications such as drug delivery and cell-based therapies.[28−30] In addition, HA-based hybrid hydrogel particulates have been developed by incorporating bioactive ceramic nanoparticles using a precipitation process to enhance their structural stability under enzymatic degradation as well as their bioactivity to promote collagen tissue growth.[31,32] Although the injectable nanocomposite system exhibits excellent biophysical properties, a high injection force resulting from the presence of nanosized ceramic fillers and nonuniform shapes and sizes of gel granules is unavoidable, thereby damaging the surrounding tissue and causing pain to the patient.[17] It is difficult for large gel particles to penetrate smoothly through thin needles, and small gel particles are attacked by enzymes because of their large surface area, resulting in poor gel persistence.[14,17,33] In some cases, uncross-linked HA is mixed as a lubricant, which further reduces the strength and persistence of the gel when the same volume of the filler is inserted.[14,17,34] Therefore, it is necessary to develop injectable gels with a uniform shape and a narrow size distribution range to achieve both excellent injectability and biomechanical strength.

To address this challenge, in our work, HA-based hybrid hydrogel microspheres prepared using a water-in-oil (w/o) method and an in situ precipitation process are introduced. By applying phase separation between the HA solutions and oil and using continuous stirring, uniformly sized highly spherical hydrogel microparticles compared to conventional crushed gels can be obtained. Furthermore, to improve their mechanical strength and biophysical stabilities, in situ precipitation of nanosized calcium phosphate (CaP) was applied to the HA microspheres. For further evaluation, we compared our spherical hydrogel constructs, the pure HA microspheres and HA-based hybrid microspheres, with conventionally granulized gel particles. The shapes and morphologies were observed, and their injectability and rheological properties were also evaluated. Furthermore, the biostability of our microsphere systems was also explored both in vitro and in vivo using enzyme solutions and magnetic resonance imaging (MRI) after subcutaneous injection, respectively. Remnant hydrogel formulations with a surrounding tissue were examined using histological stainings to verify their biocompatible and biostable properties. This comparative analysis is meaningful because there are limited reports comparing gel microspheres prepared by the w/o method with crushed gel particles, and the introduction of in situ precipitation in their production is a novel approach.

Results and Discussion

Morphology of Gel Microspheres and Crushed Gels

The HA microspheres and HA crushed gels are referred to as Sphere and Crush, respectively, and the CaP-precipitated hybrid gels are referred to as Sphere-P and Crush-P, respectively.

The appearance of the gel was examined using optical microscopy (OM). Sphere exhibited a completely spherical shape (Figure B) since HA solution is hydrophilic, while olive oil is hydrophobic; therefore, HA droplets were formed in the oil during rotation and were cross-linked. The size of the particles was adjustable by varying the rotational speed of the HA and oil emulsion. The diameter decreased from 896 ± 174 μm to 433 ± 124, 223 ± 69, and 97 ± 17 μm, as the rotational speed was increased from 200 rpm to 400, 800, and 1500 rpm. In all cases, the gel was formed into a perfectly spherical shape, and the particle size showed a normal distribution (Figure S1).

Figure 1

(A) Schematic illustration of fabrication of HA microspheres and crushed gel. (B) Optical images and (C) the size distribution of pure gel microspheres and crushed gels as well as each gel with precipitation.

(A) Schematic illustration of fabrication of pan class="Chemical">HA microspheres and crushed gel. (B) Optical images and (C) the size distribution of pure gel microspheres and crushed gels as well as each gel with precipitation.

The particle size of Crush was much more varied (Figure B). The gel was physically crushed by a homogenizer; however, it did not break into uniform shapes, and small particles of several tens of micrometers and large particles of several hundreds of micrometers coexisted. The average size of the particles was depended on the rotational speed of the homogenizer; however, its distribution was difficult to control. The gels had a wide size distribution with high average size by a slow rotational speed; however, the average size was excessively decreased with high speed; 265 ± 190, 222 ± 172, and 77 ± 74 μm, as the rotational speed was increased from 2000 rpm to 5000 and 8000 rpm (Figure S2).

Comparing Sphere and Crush with similar averages, Crush had a much wider distribution than that for Sphere; average diameters were 222 ± 172 μm for Crush and 223 ± 69 μm for Sphere (Figure C). The range of the particle size distribution is important because it affects the extrusion force required for the injection.[14,33] Not only the spherical shape but also the narrow distribution of Sphere makes this material more appropriate for injectable applications than Crush. Crush-P and Sphere-P, in which CaP nanoparticles were precipitated, were also examined, and their appearances were similar to those of the pure Crush and Sphere samples, respectively (Figure B). The particle sizes were 207 ± 146 and 211 ± 83 μm, respectively, similar to the each pure gels (Figure C).

Characteristics of Gel Microspheres

SEM images of the surfaces of the microspheres are presented in Figure A. Both Sphere and pan class="Chemical">Sphere-P were spherical, without distortion after the precipitation. At high magnification, Sphere exhibited a smooth surface, whereas pan class="Chemical">Sphere-P exhibited a nanoroughened surface because of the CaP precipitates.

Figure 2

(A)–(D) Characteristics of pure gel microspheres and those after precipitation. (A) SEM images, (B) FT-IR spectra, (C) XRD patterns, and (D) TGA curves of pure and precipitated HA microspheres.

FT-IR spectra of all the gel groups and HA powder are presented in Figure B. The characteristic peaks of HA appear at 1034, 1405, and 1607 cm–1. These peaks correspond to C–O stretching, alkyl groups (−CH), and carboxyl groups (−COOH), respectively. The broad peaks near 2891 and 3279 cm–1 correspond to the alkyl group (−CH) and hydroxyl group (−OH), respectively.[35] These peaks were observed in the HA powder and were also clearly visible for all four types of gel samples. Therefore, the gel microspheres were formed well without residual oil or degradation of HA. For the precipitated gels, a peak corresponding to PO43– appeared at 556 cm–1, indicating the successful formation of CaP.[36]

The components of the precipitates were analyzed using XRD (Figure C). No peaks were observed for the pure gel. Small and broad peaks were observed for Sphere-P because the crystallinity of the precipitates was low, and the nanosized precipitates coexisted between polymer chains. Peaks appeared at approximately 26 and 32°, corresponding to CaP; thus, the composition of the precipitates was expected to be CaP. To more accurately confirm the composition, heat treatment was performed at 1250 °C for 30 min. The peaks were detached, and their compositions were determined to be hydroxyapatite and tricalcium phosphate.

The amounts of the precipitates were analyzed using TGA (Figure D). All the TGA curves show similar tendencies. The initial weight loss was due to the evaporation of water and solvent, and the weight decreased until 500 °C because of the degradation of HA. After that, all the samples exhibited a residual mass. For Crush and Sphere, residues such as sodium carbonate remained at approximately 10%; however, Crush-P and Sphere-P exhibited a residual mass of approximately 30%. Thus, the precipitate content was approximately 20 wt % regardless of the type of gel.

Characterization of Cross-Linking Density

The 1H-NMR spectra used to characterize the cross-linking density of Crush and Sphere are presented in Figure A. For both Crush and Sphere, peaks appeared at 1.66 and 2.05 ppm, corresponding to protons of the alkyl groups in the cross-linking agent, 1,4-butanediol diglycidyl ether (BDDE) and protons of the N-acetyl groups in HA, respectively (Figure A,B). The degree of cross-linking was determined by comparing the relative peak integrals, and Crush and Sphere exhibited cross-linking densities of 21.22 and 28.09%, respectively (Figure C). Thus, the cross-linking density of Sphere was approximately 32% greater than that of Crush.

Figure 3

(A)–(D) Characterization of cross-linking densities of gel microspheres and crushed gels: (A) 1H-NMR spectra of crushed gels and gel microspheres, (B) schematic images of cross-linking of HA, (C) degree of cross-linking of both gels, and (D) swelling ratio and water contents of the gels. The results are presented as the mean ± SD (n = 3).

The swelling ratio and water content of the gels were also examined (Figure D). Prior to comparison between Sphere and Crush, the analyses of the crushed gels and uncrushed bulk gels showed no significant difference, which means that the crushing process did not affect the wettability of the gels (Table S1). The swelling ratio of Sphere was 23%, which was lower than that of Crush (35%), indicating that Sphere had a higher cross-linking density than Crush. Because more cross-linking prevents the gel from absorbing much water, there is an inverse relationship between the swelling ratio and cross-linking density. In addition, the water contents of the precipitated gel were 96.24% for Crush-P and 94.99% for Sphere-P, representing reductions of approximately 1% from those of the pure gels because the CaP precipitates not only acted as a strengthening agent but also improved the bonding of the polymer chains.

Mechanical Properties of Gel Microspheres and Crushed Gels

The viscoelastic behaviors of the spherical and crushed gels were investigated using a rheometer, as shown in Figure A. The storage moduli (G′) of all the samples were shown to be more dominant than the loss moduli (G″) over the full range of angular frequency (data not shown), indicating that highly elastic behaviors were achieved. In addition, Sphere exhibited a much higher G′ than Crush over the full range of frequency. For frequency sweeps, G′ at 6.3 rad/s was 735 Pa for Crush and more than doubled to 1764 Pa for Sphere because of the enhanced cross-linking. Furthermore, Crush showed liquid-like behavior where G′ varies with frequency, whereas Sphere exhibited a more solid-like and stiff characteristic with constant G′ regardless of the frequency. The G′ values of the gels were further increased by approximately 40% after precipitation to 1074 and 2381 Pa, respectively, and Crush-P exhibited predominantly elastic behaviors. In strain sweep, Sphere also exhibited higher G′ than Crush over the entire strain range. Furthermore, the linear viscoelastic region (LVR) was longer for precipitated gels, that is, the yield point of deformation (the critical strain) was higher than pure gel samples. This is because the CaP nanoparticles, which had formed homogeneously between the HA chains, entangled the HA chains, and the gel becomes ductile and can withstand large deformation. The complex viscosity (η*), which means the total ability of a material to withstand deformation, was calculated by considering the G′ and G″. Although all specimens showed shear thinning, the η* appeared in the same order as Sphere-P, Sphere, Crush-P, and Crush at all shear rates (Figure S3).

Figure 4

(A)–(C) Mechanical and injectional properties of crushed gels and gel microspheres and each gel with precipitation. (A) Rheological behavior showing storage modulus for frequency sweep and (B) injectability of each gel. (C) Relationship between injectability and storage modulus graph of HA gels. The results are presented as the mean ± SD (n = 3).

Thus, the stiffness was remarkably increased for Sphere, especially after precipitation. In conclusion, the rheological properties of Sphere were similar to those reported for HA dermis fillers, indicating its appropriateness for injectable hydrogels in therapeutic applications.[15,42,43] Furthermore, Sphere-P exhibited improved properties and is thus suitable for the treatment of deep wrinkles or severe skin deformities as well as the treatment of knee joints with high load bearing.

Injectability of Gel Microspheres and Crushed Gels

The injectability was analyzed by measuring the injection force (Figure B). Crush required an injection force of approximately 20 N, whereas Sphere was easily injected using an injection force of approximately 5 N, demonstrating its improved injectability. For Crush, large particles occasionally blocked the needles and increased the injection force. However, Sphere passed through the needle without clogging because of its relatively narrow size distribution and uniform spherical span class="Chemical">hape.

The effect of precipitate particles on the injection force was also evaluated. For Crushpan class="Chemical">-P, the gels became stiffened after precipitation, resulting in an increased injection force. pan class="Chemical">Sphere-P was also stiffened, resulting in an increase of the injection force; however, it was still released below 10 N without a rapid increase in force.

The relationship between G′ and injection force is summarized in Figure C. G′ was determined at a strain of 1% and a frequency of 6.3 rad/s, and the injection force was averaged over a linear 2 mm section.[44] Crush was prepared using various amounts of BDDE, and there was an exponential relationship between G′ and the injection force. Also, the Sphere group was confirmed to exhibit a low injection force and high G′. In general, HA particles with higher G′ values are more difficult to inject, even if the gel particles are very small because it is difficult to pass a stiffened gel through a needle.[17] However, despite high G′, both Sphere and Sphere-P required significantly lower injection forces due to their uniform size and shape compared to crushed gels. Overall, compared with the conventional crushed products, Sphere-P exhibited improved mechanical properties owing to the combination of its high G′ and high injectability.

In Vitro and In Vivo Degradation Behaviors

The biodegradability of the gels was confirmed by analyzing the degradation behavior both in vitro and in vivo. For an in vitro analysis, in all the cases, the residual weight of the gel increased in the early stage because the initial degradation loosened the polymer chains and increased water absorption (Figure ).[45] After that, further degradation occurred, and the mass decreased. After 24 h, the residual weight of Crush remained at approximately 30%; however, that of Sphere was almost 100%, indicating that Sphere was more stable than Crush. The first explanation for this result is that the high cross-linking reduced the swelling ratio and increased the enzymatic resistance of the HA chain against hyaluronidase.[46] Furthermore, Sphere is likely to be longer lasting because of its relatively uniform size and small surface area, whereas Crush is a mixture of large and small particles and has a large surface area, which can be easily attacked by external enzymes. In addition, the precipitated gels exhibited higher stability than the pure gels because CaP particles acted as cross-linking agents between HA chains to enhanced persistence. Therefore, Sphere-P was the most stable under in vitro physiological conditions.

Figure 5

In vitro degradation behaviors of the gel by hyaluronidase at 7 UI/mL. The results are presented as the mean ± SD (n = 3).

In vitro degradation bepan class="Chemical">haviors of the gel by hyaluronidase at 7 UI/mL. The results are presented as the mean ± SD (n = 3).

Based on the demonstrated degradation resistance properties, the in vivo volumetric persistence of the Sphere groups under the skin was also evaluated using MRI and compared with that of Crush. Figure A presents cross-sectional images of the center of the injected gel as a function of time. The white part corresponds to the residual gel and surrounding tissues because MRI detects moisture. Sphere-P appeared to be maintained more, followed by Sphere; however, Crush appeared to be more widespread and less remained. After 8 weeks, Sphere-P became darker indicating its water content was relatively low compared with that of the pure gel. After 28 weeks, Sphere-P maintained a significantly high volume, followed by Sphere, and Sphere was less diffused and more rounded than Crush.

Figure 6

(A, B) In vivo degradation behaviors over time: (A) MRI images of crushed gels, gel microspheres, and gel microspheres with precipitation at 0, 8, 16, and 28 weeks after injection (scale bars: 1 mm). (B) Remaining volume of each gel as a function of time until 28 weeks. The results are presented as the mean ± SD (n = 5).

(A, B) In vivo degradation bepan class="Chemical">haviors over time: (A) MRI images of crushed gels, gel microspheres, and gel microspheres with precipitation at 0, 8, 16, and 28 weeks after injection (scale bars: 1 mm). (B) Remaining volume of each gel as a function of time until 28 weeks. The results are presented as the mean ± SD (n = 5).

The entire volume was measured by calculating the area of fillers in 2D images and stacking them (Figure B). Crush showed a significant decrease compared with Sphere groups at 1 week, and there was a significant difference between the three groups after 8 weeks. When all three groups were expressed as a spline-β line, the volume of residual gel was decreased linearly; Crush and Sphere exhibited a sharp decrease between 4 and 8 weeks, and Sphere-P showed a rapid decrease between 16 and 20 weeks. At 28 weeks, Sphere-P, Sphere, and Crush exhibited residual gel volumes of 45, 36, and 23%, respectively. That is, in addition to the precipitation, only pure Sphere had significantly improved stability and maintained a round shape of the gel bundle better than pure Crush, which is consistent with the previous evaluations.

In Vivo Histological Observation

The histology was analyzed after staining to evaluate the bioactivity and tissue regeneration. In hematoxylin and eosin (H&E) staining, the blue color corresponds to the remaining gel, and the purple color corresponds to the soft tissue (Figure A,B). The white part corresponds to the empty space created by the partial removal of the gel during the process of dehydration and paraffin treatment, which is a pretreatment process used for histological staining. For Crush, the gel was fully depressed, whereas for Sphere, an extensive fibrous tissue was observed between the gels (Figure A). Sphere retained its original shape from an external force because the stiff gels exhibited high resistance to deformation. Furthermore, spherical-shaped particles are in point contact with other particles, while irregular-shaped particles make surface contact with other particles, reducing the volume of pores. Therefore, Sphere resulted in better volume restoration than that of Crush. In addition, the tissue could infiltrate and regenerate between the gels. This is beneficial in which the injected gel maintains its volume well, the large contact area reduces foreign body reactions, and the regenerated tissue maintains the space even after the degradation of the gel. Furthermore, more regeneration was observed for Sphere-P, and this difference was more apparent at high magnification (Figure B). It is difficult to find any regenerated tissue or cells in Crush; however, regenerated tissue with fibroblasts was formed between the gels in Sphere. In addition, Sphere-P showed improved tissue regeneration with more fibroblasts, the purple dots. Red dots (endothelium) are also observed in a rounded collection, which indicates that the blood vessels were formed after maturation of the new tissue. The fibroblast densities are presented in Figure C, and Sphere showed a significant increase compared with that of Crush. Also, Sphere-P was further increased compared with that of the other two specimens.

Figure 7

(A)–(G) Histologically stained tissues injected with crushed gels, gel microspheres, and gel microspheres with precipitation after 12 weeks of injection. (A, B) H&E stained images and (C) density of fibroblasts in ingrowth tissue. (D) Masson’s trichrome-stained images and (E) collagen area in ingrowth tissue. (F) Masson’s trichrome-stained images and (G) dermal thickness in dermis. The results are presented as the mean ± SD (n = 3, **P < 0.01). (Scale bars for (A) = 1 mm, for (B) = 50 μm, and for (C, D) = 120 μm).

The promotion of tissue formation for Sphere-P was affected by two reasons. First, the adhesion and proliferation of fibroblasts are enhanced by the increase in stiffness of the hydrogel.[47,48] Because stiffer gel maintains its shape well in the body and provides a large space for cell attachment, fibroblasts attach well forming the collagen and ECM for dermal tissue. Thereby, the proliferation of fibroblasts and tissue regeneration were improved. Furthermore, the CaP nanoparticles induced significant fibroblast cell migration from the surrounding tissue and stimulated the biological response for the generation of collagen and ECM production.[49,50] The mechanism can be explained by the effect of calcium ions (Ca2+). The Ca2+ signal activated the biological function of fibroblasts by enhancing the activation of SMAD, which is the main signal transducers for receptors of the transforming growth factor beta (TGF-β).[51] It has been reported that gene expression including SMAD2 and SMAD3 were higher in the hyaluronic acid (HA) composites containing CaP than pure HA.[52] Therefore, in Sphere-P, the released Ca2+ promoted expression of a TGF-containing growth factor through a SMAD pathway, resulting in an increased fibroblast density and tissue formation.

In addition, immunohistochemical staining with an anti-CD68 antibody was performed to confirm the immune response. Macrophages should be stained brown; however, it was difficult to see macrophages in all the groups, and there was no difference from normal tissues. For Sphere-P, the many blue dots are indicating nonimmune cells, but the normal cells were detected; thus, they can be regarded as fibroblasts. Quantitative analysis revealed no difference in the expression of CD68 among the three experimental groups (Figure S4).

Masson’s trichrome staining was also performed to confirm the maturity of the tissue, as shown in Figure D,F. Collagen appears blue; therefore, a higher blue ratio indicates a more matured fibrous tissue. Crush had a little tissue and was reddish, whereas Sphere was partially blue, and Sphere-P was mostly blue in an ingrowth tissue (Figure D). Sphere-P was more biocompatible because of bioactive CaP and mechanical strengthening; therefore, the fibroblasts could stretch for a robust collagen synthesis. There was a significant difference in the area of collagen in the generated tissue among the three groups, as observed in Figure E.

Biological responses on the dermis were also examined. The dermal thickness after filler injection was analyzed (Figure F). When the filler was inserted, the upper dermis tissue was stretched and thinned. Using the bioactive filler, the dermis was stimulated, and the dermal thickness approached the original state. The thicknesses of Crush and Sphere were similar (approximately 100 μm), whereas that of Sphere-P was approximately 180 μm (Figure G). The references indicate that the dermal thickness of the nude mouse is approximately 200 μm;[53] therefore, Sphere-P almost recovered its original thickness. The CaP particles promoted cell growth and tissue formation. In conclusion, we expect Sphere-P hydrogels, which possess excellent biostability and biocompatibility, to be suitable for injectable filler clinical applications.

Conclusions

In this study, HA microspheres were successfully fabricated using a w/o emulsion process and were compared with conventional crushed gels cross-linked in bulk. The Sphere gels were fabricated with a relatively uniform size and a completely spherical shape, and their size was easily controllable by adjusting the rotational speed. Sphere had enhanced cross-linking density compared with Crush, resulting in a lower swelling ratio and improved mechanical properties. Furthermore, the injectability was significantly increased due to the narrow size distribution and spherical shape. The biostability was also improved because of the higher cross-linking density and reduced surface area resulting from the relatively uniform size. This tendency was further improved after in situ CaP precipitation, which resulted in a significant increase in strength and biostability with only a negligible increase in the injection force. These effects were confirmed in in vivo tests. Sphere was significantly more biostable than Crush, and the shape of the filler was well retained. In addition, for Sphere, new tissues grew by infiltration between the HA gels, and the area of collagen in ingrowth tissues also increased. For Sphere-P, the CaP nanoparticles stimulated cell growth and tissue formation, resulting in the formation of more matured tissues and having a positive effect on the dermis. Therefore, Sphere shows better potential for injectable hydrogel applications than Crush in terms of the rheological properties, bioactivity, and biostability, and these properties were further improved after CaP precipitation.

Experimental Section

Materials

Biograde HA sodium salt with a molecular weight of 1.8–2.5 MDa and a purity of 99.84 + % produced by fermentation of Streptococcus zooepidemicus was purchased from SK Bioland, Cheonan, Korea. Phosphate-buffered saline (PBS) was obtained from Welgene, Gyeongsangbuk-do, Korea, and zoletil was supplied from Virbac, Carros, France. Rompun was obtained from Bayer, Leverkusen, Germany, and the anti-CD68 antibody was supplied form Biorbyt, Cambridge, UK. All the other chemicals were purchased from Sigma-Aldrich, MO, USA.

Fabrication of Gel Microspheres

The HA microspheres were prepared using a w/o emulsion technique. First, the HA powders were gently stirred in 0.2 M sodium hydroxide (NaOH) solution at 10 °C overnight to prepare a 10% (w/v) HA solution. The cross-linking agent, BDDE, was mixed with the prepared HA solution at 2% (v/v) and emulsified in olive oil at 10% (v/v). After 1 h, the stirring speed was fixed at the desired levels, that is, 200, 400, 800, and 1500 rpm. The cross-linking was performed at 25 °C for 4 days with stirring because long-term cross-linking at low temperatures (below 37 °C) has been identified to be more beneficial in enhancing physical properties, than at conventional condition (around 37 °C or higher).[54]

The cross-linked HA microspheres were vacuum filtered using a Buchner funnel with a quantitative filter paper (Advantec, CA, USA; pore 1 μm, thickness 0.22 mm) to remove olive oil. The filter paper was placed on the Buchner funnel above a triangular filtering flask, and a rotary pump was connected to the flask to hold the vacuum. The washing solution was repeatedly poured into the filter paper in a volume of 100 mL at a time. The washing solution was initially 70% acetone aqueous solution, gradually decreasing the concentration, and finally, distilled water (DW) and PBS were used. After that, 2 g of microspheres were immersed in 10 mL of PBS for 2 h, and PBS was replaced every 30 min for complete washing. Then, the microspheres were swollen in PBS at 37 °C for 24 h. Swollen microspheres were separated from PBS using gravity filtering and were used in the experiments.

To prepare the hybrid microspheres, CaP nanoparticles were precipitated inside the gel. After filtering and washing the residual oil, the pure HA microspheres were immersed in 0.145 M calcium chloride (CaCl2) and 0.087 M phosphoric acid (H3PO4) solution for 2 h. They were then immersed in 10% (v/v) ammonium hydroxide (NH4OH) solution for 30 min. The residue was removed by washing with PBS, and the hybrid microspheres were fully swollen in PBS at 37 °C for 24 h.

A schematic illustration of the fabrication process of the gel microspheres and crushed gels is presented in Figure A.

Fabrication of Crushed Gels

The crushed gels were fabricated by preparing a bulk HA gel and crushing it. After mixing BDDE and 10% (w/v) HA solutions at 2% (v/v), 1 mL of each solution was sealed in a polyethylene mold and cross-linked at 25 °C for 4 days. The cross-linked bulk gel was washed with PBS for 2 h to remove residual BDDE and then swollen at 37 °C for 24 h. After the surface of the swollen gel was wiped, it was crushed for 4 min at 2000, 5000, and 8000 rpm using a homogenizer (T18 digital ULTRA-TURRAX, IKA, Staufen, Germany).

For the hybrid crushed gels, the same process was performed on the bulk hydrogel. CaP nanoparticles were precipitated inside the gel. After washing, the bulk HA gel was immersed in 0.145 M CaCl2 and 0.087 M H3PO4 solutions for 2 h and then in 10% (v/v) NH4OH solution for 30 min, in sequence. The residue was removed by washing with PBS, and the bulk hybrid gels were fully swollen in PBS at 37 °C for 24 h and then crushed under the same conditions.

Characterization of HA Gel Particles

The swollen gels were observed using optical microscopy (OM; Dimis-M, Siwon Optical Technology, Gyeonggi-do, Korea) after staining with 3% (w/v) pan class="Chemical">alcian blue in 1% (v/v) pan class="Chemical">acetic acid solution. The sizes of all gels were analyzed using Image J program (National Institutes of Health, MD, USA).

The gels were also cpan class="Chemical">haracterized using Fourier-transform infrared (FT-IR) spn>ectroscopy (Nicolet iS50, Thermo Fisher Scientific, MA, USA) for wavenumbers ranging from 400 to 4000 cm–1 with an average of 32 scans after lyophilization.

The cross-linking density was determined using 1H-NMR spectroscopy (AVANCE-500, Bruker, MA, USA). Crush and Sphere were dissolved in deuterium oxide after treatment with 0.01 mg/mL (7 UI/mL) hyaluronidase solution for 5 days and then dried.[55,56] The cross-linking density was calculated by comparing the ratio of the relative peak integrals of 2.05 ppm; the protons of the N-acetyl group in HA, to 1.66 ppm, the protons of the alkyl group in BDDE.

Characterization of CaP Nanoparticles in Sphere-P

The properties of CaP nanoparticles were evaluated after drying the gel particles at room temperature. The surface of Sphere-P was characterized using field-emission scanning electron microscopy (FE-SEM; MERLIN Compact, Carl Zeiss Inc., Oberkochen, Germany) under an accelerating voltage of 2 kV. The crystalline phases were characterized using X-ray diffraction (XRD; D8-Advance, Bruker Co., Karlsruhe, Germany) over the scanning range of 20–60°.

The CaP content was quantified using a thermogravimetric analysis (TGA; simultaneous DTA/TGA analyzer, TA Instruments, DE, USA). Before the analysis, the gels were completely dried in air and heated to 1000 °C at a rate of 10 °C/min under air condition.

Mechanical Properties and Injectability

The rheological behaviors of all the gels were analyzed using a rheometer (Discover HR-2, TA Instruments, DE, USA) with a parallel-plate geometry. All the swollen gels were prepared in a syringe in a volume of 1 mL and dispensed onto the bottom plate, and then the top plate with a 20 mm diameter was plated in a gap of 2.8 mm. Frequency sweep tests were performed in the frequency range of 0.1–100 rad/s at a strain of 0.1%. Strain sweep tests were performed over the strain range of 0.01–100% at a frequency of 1 Hz.

The injection force was measured using a universal testing machine (Instron 3343, Instron, MA, USA) with a 50 N-capacity load cell. A 1 mL syringe was filled with 0.8 mL of each gel, and a 27-gauge needle was inserted. After tpan class="Chemical">hat, a compression force was applied vertically at a crosshead speed of 10 mm/min, and the force when the gel was released through the needle was measured.

A plot of the storage modulus (G′) as a function of the injection force was obtained for various pan class="Chemical">BDDE concentrations of the crushed gels (1, 2, 4, 6, 8, and 16% (v/v)).

Swelling Ratio and Degradation Behavior

The swelling ratio was calculated from the weights of the swollen and dried gel. All the gels were swollen in PBS solution at 37 °C for 24 h, and the swollen weight (ws) was measured. They were then dried in a vacuum oven and weighed to obtain the dried weight (wd). The swelling ratio and water content of each gel were calculated using the following equations:

The degradation bepan class="Chemical">havior was assessed by measuring the weight of each gel over time in the hyaluronidase solution. Each gel was immersed in hyaluronidase solution diluted to 0.01 mg/mL (7 UI/mL) in pan class="Chemical">PBS at 37 °C. The initial weight (wi) was measured at the beginning, and the remaining weight (wr) was measured at each time point. The remaining weight of each gel was calculated using the following equation:

In Vivo Assessment

Six-week-old female BALB/c nude mice were obtained from a commercial vendor (Orient Bio Inc., Seongnam, Korea). In total, seven mice were used (four for the MRI analysis and three for the histological analysis), and three types of injections were performed randomly for each mouse four times: (i) Crush, (ii) Sphere, and (iii) Sphere-P (n = 5 per group for MRI, and n = 3 per group for histology). After feeding the mice a standard diet and allowing them to rest for 2 weeks, the mice were anesthetized via an intraperitoneal injection of zoletil (30 mg/kg) and rompun (5 mg/kg). After the anesthetic was administered, 200 μL of each gel was injected into the back of each mouse between the panniculus adiposus layer and panniculus carnosus, with a total of 4 injections per mouse (Figure S5). The in vivo animal experiment was approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC protocol no. 18-0014).

Volumetric and Histological Analyses

All the groups were examined using the MRI volumetric analysis under isoflurane/O2 (1.5% isoflurane, 1.0 L/min O2) immediately after the injection and after the first week and every 4 weeks up to 28 weeks. A magnetic resonance scanner (Agilent 9.4 T/160AS, Agilent Technologies, CA, USA) and a 1H surface coil (Millipede Coil, Agilent Technologies, CA, USA) were used for the MRI analysis. Both T2-weighted axial and coronal images were obtained. The slice thicknesses of the axial and coronal image were 0.3 and 1.0 mm, respectively. The axial T2-weighted image was used for the volumetric analysis performed using Image J program.

For histological observation, the injected gels and surrounding tissue were extracted at 12 weeks. They were immediately fixed in 10% formalin for 24 h and then gradually dehydrated with ethanol and embedded in paraffin. The paraffin blocks were sectioned into 4 μm thicknesses. The slices were stained with hematoxylin and eosin (H&E) and Masson’s trichrome to visualize the remaining gel and infiltrated as well as the surrounding tissue. The analyses of the collagen area and dermal thickness were performed using Image J program.

Statistical Analysis

All the assays were performed with a minimum of n = 3 per group. All the quantitative variables are presented as means ± standard deviations. The data were analyzed using the Statistical Package for the Social Sciences (SPSS) (IBM, NY, USA), statistical software program. For multiple comparisons between the groups, a one-way analysis of variance (ANOVA) and a Tukey’s post-hoc analysis were used. The statistical significance was set at **p < 0.01.