Article pubs.acs.org/Biomac
Strong and Biostable Hyaluronic Acid−Calcium Phosphate Nanocomposite Hydrogel via in Situ Precipitation Process Seol-Ha Jeong,† Young-Hag Koh,‡ Suk-Wha Kim,§ Ji-Ung Park,∥ Hyoun-Ee Kim,†,⊥ and Juha Song*,†,⊥ †
Department of Materials Science and Engineering, Seoul National University, Seoul 151-742, Korea School of Biomedical Engineering, Korea University, Seoul 136-703, Korea § Department of Reconstructive and Plastic Surgery, Seoul National University Hospital, Seoul 110−744, Korea ∥ Department of Plastic and Reconstructive Surgery, Seoul National University Boramae Hospital, Seoul 156-707, Korea ⊥ Biomedical Implant Convergence Research Center, Advanced Institutes of Convergence Technology, Suwon 443-270, Korea ‡
S Supporting Information *
ABSTRACT: Hyaluronic acid (HAc) hydrogel exhibits excellent biocompatibility, but it has limited biomedical application due to its poor biomechanical properties as well as too-fast enzymatic degradation. In this study, we have developed an in situ precipitation process for the fabrication of a HAc-calcium phosphate nanocomposite hydrogel, after the formation of the glycidyl methacrylate-conjugated HAc (GMHA) hydrogels via photo-cross-linking, to improve the mechanical and biological properties under physiological conditions. In particular, our process facilitates the rapid incorporation of calcium phosphate (CaP) nanoparticles of uniform size and with minimal agglomeration into a polymer matrix, homogeneously. Compared with pure HAc, the nanocomposite hydrogels exhibit improved mechanical behavior. Specifically, the shear modulus is improved by a factor of 4. The biostability of the nanocomposite hydrogel was also significantly improved compared with that of pure HAc hydrogels under both in vitro and in vivo conditions. bioactive glass22 and calcium phosphate (CaP)23 is one of the most promising options for improving mechanical stability as well as biofunctionality. For example, blending nano silica as a mechanical reinforcement improved the mechanical strength of HAc chains.24 In another study, nano CaP was incorporated into a functionalized HAc matrix for the production of very tough composite hydrogels.25 The incorporation of CaP nanoparticles enhanced the mechanical properties of the composite networks by providing a robust physical registration between nanoparticles and functionalized groups of the HAc; however, those specimens were prepared by simple physical mixing of the inorganic particles with the HAc.26 This approach is simple, but inhomogeneous distribution of the nanoparticles or outright agglomeration during mixing is one of the major concerns.27 This often results in morphological and compositional inhomogeneity of the nanoparticles, which detracts from the sought mechanical improvements.23,28−31 In this study, we have employed an in situ precipitation process for the production of HAc/CaP composite hydrogels. In situ precipitation has been shown to be effective in generating uniformly distributed CaP nanoparticles in alginate-based biofilms32 and chitosan hydrogels.32,33 The
1. INTRODUCTION Hyaluronic acid (HAc), a well-known natural polysaccharide material, is a major intracellular component of connective tissues, where it plays an important role in lubrication, cell differentiation, and cell growth.1,2 Because of its ability to maintain a hydrated environment for cell infiltration and its nonimmunogenic, biocompatible, and biodegradable nature, HAc-based scaffolds have been extensively investigated and used for a wide variety of biomedical applications including the treatment of arthritis,3 ophthalmic surgery,4,5 drug delivery,6 tissue engineering such as cartilage7 and bone repair as well as regeneration,8 and dermal skin tissue augmentation;9,10 however, linear and soluble HAc molecules in the natural state are not suitable for scaffolds because of their poor biomechanical properties and too fast in vivo degradation through enzymatic reaction with hyaluronidase.11−13 Therefore, various cross-linking methods through chemical modification of functional groups have been developed to improve the mechanical properties and biostability of HAc.14−17 Still, applications of HAc hydrogels with various cross-linking systems are limited due to its short residence time and lack of mechanical integrity. Recently, composite systems based on HAc hydrogels have been proposed, bringing about further mechanical and biological improvements by introducing different polymeric materials or bioactive nanoparticles.18−21 Among these methods, incorporating inorganic particles such as © XXXX American Chemical Society
Received: November 17, 2015 Revised: February 1, 2016
A
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules Scheme 1. Schematic Diagram of the Fabrication of pHAc-CaP Hydrogels via in Situ Precipitation
extracted and dried in air for 2 days. The phase of the precipitated calcium phosphate was analyzed using an X-ray diffractometer (XRD, D8-Advance, Bruker, Germany). Samples of three different conditions were prepared: pure HAc hydrogel, pHAc-30 wt % CaP before heat treatment, and pHAc-30 wt % CaP after heat treatment at 1100 °C for 5 min. XRD data were obtained from 20 to 60° (2θ) using Cu Kα radiation with a scan rate of 1°/min. The actual CaP content was quantified by thermogravimetric analysis (TGA, Simultaneous DTA/ TGA analyzer, TA Instruments, USA), which showed good agreement with the theoretical content. Prior to the analysis, all hydrogel samples were completely dried in air and heated to 1100 °C at the rate of 10 °C/min under an N2 atmosphere. (See Supplemental Tables S1 and S2 for further details.) 2.3. Mechanical Properties of Hydrogels. The rheological behavior of the hydrogels was assessed using a controlled strain rheometer (ARES, Rheometric Scientific, USA). All hydrogels were prepared with a diameter of 25 mm and a thickness of 2 mm. Frequency sweeps were carried out in the angular frequency range of 0.1 to 100 rad/s at strain (1%) in the linear region to measure the mechanical properties including the storage (G′) and loss (G″) moduli and the complex viscosity. 2.4. Swelling Properties and in Vitro Behaviors of Hydrogels. The equilibrium swelling ratio of the hydrogels was evaluated in a PBS solution at 37 °C. The hydrogels were dried in a vacuum oven and weighed to obtain the initial weight (Wi). They were then immersed in PBS for 24 h and reweighed to obtain the hydrated weight (Wh). The swelling ability of the hydrogels was calculated according to the equation
mechanical and physical properties and biological performance of the hydrogel systems were markedly enhanced through uniform distribution of the CaP nanoparticles. This composite system has great potential for regenerative tissue augmentation and other tissue engineering applications.
2. MATERIALS AND METHODS 2.1. Fabrication of Nanocomposite Hydrogels through in Situ Precipitation. The schematic diagram of the in situ precipitation process for fabricating HAc-CaP nanocomposite hydrogels (pHAcCaP) is illustrated in Scheme 1. First, HAc hydrogels were photo-cross-linked using a previously proposed protocol.34 In brief, to create glycidyl methacrylatehyaluronic acid (GMHA), 1% w/v HAc with a molecular weight of 1.8 to 2.5 MDa (Bioland, Seoul, Korea) was dissolved in 100 mL of phosphate-buffered saline (PBS). Then, 2.2% v/v of triethylamine, glycidyl methacrylate, and 2.2% w/v tetrabutylammonium bromide were added to the HAc solution in sequence. After stirring overnight at room temperature, the GMHA conjugates in solution were precipitated by the addition of acetone and cleansed with distilled water to remove excess reactants. To cross-link the hydrogels, GMHA was redissolved in distilled water and exposed to UV light for 15 min with a photoinitiator (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, Irgacure 2959). Subsequently, the hydrogels were immersed overnight in a solution of calcium chloride and phosphoric acid with a Ca/P ratio of 1.67 (pH < 7). The amounts of these two reactants were varied to yield CaP content ranging from 10 to 40 wt % (see Supplemental Table S1 for further details), and the products were called ‘pHAc-10, 20, 30, or 40 wt % CaP’. This procedure is henceforth referred to as the “ion permeation process”. Subsequently, the Ca/Prich HAc hydrogels were dipped in 15% ammonium hydroxide solution for 3 h to precipitate calcium phosphate within the hydrogels. This process is the “in situ precipitation process.” The hydrogels were dialyzed in a PBS solution for 48 h to remove the remnants of the ammonium hydroxide solution. The composite hydrogels prepared by physical mixing (mHAc-CaP) were also based on the aforementioned cross-linking system. 2.2. Characterization of pHAc-CaP Hydrogels. The morphology of the nanocomposite hydrogels was observed using a fieldemission scanning electron microscopy (FE-SEM, SUPRA 55VP, Carl Zeiss, Germany). Prior to the observation, the samples were lyophilized overnight to maintain the structure of the samples. The structure and chemical composition of the precipitated nanoparticles in the hydrogels were evaluated by transmission electron microscopy (TEM, Tecnai F20, FEI, USA). 400-mesh copper grids were placed in the nanocomposite hydrogels during the fabrication process and were
swelling ratio (g/g) = (Wh − Wi /Wi ) where Wi and Wh represent the initial dried weight and the hydrated weight, respectively. In vitro degradation tests for the hydrogels were performed in a PBS solution at 37 °C with hyaluronidase (Type I−S, lyophilized powder, 400−1000 UI/mg solid, Sigma Co., Korea) at a concentration of 500 μg/mL (200−500 UI/mL). The degradation rates for the hydrogels were investigated by immersing them in the hyaluronidase solution for predetermined periods of time and measuring the weight changes calculated as
remaining weight ratio (%) = (Wr /Wi ) × 100 where Wi and Wr represent the initial weight and the remaining weight, respectively. A fibroblast cell line, L929 (derivative of strain L, Mus musculus, mouse), was used to assess the cellular responses on the hydrogels. Prior to cell seeding, the samples were washed with 70% ethanol and B
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules PBS solution and sterilized on a clean bench under UV irradiation. The preincubated cells were seeded on the hydrogels at densities of 5 × 104 cells/mL for the cell attachment test and at 2 × 104 cells/mL for the cell proliferation test. The cells were cultured in an alpha-minimum essential medium (α-MEM, Welgene, Korea) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin in a humidified incubator with 5% CO2 at 37 °C. The cell attachments were visualized using confocal laser scanning microscopy (CLSM, FluoView FV1000, Olympus, Japan) after culturing for 3 days. Prior to visualization, the cultured cells on the samples were fixed in 4% paraformaldehyde in PBS for 10 min, washed 3 times with PBS solution, and then permeabilized with 0.1% Trion X-100 in PBS for 5 min and 1% BSA in PBS for 30 min, respectively. Subsequently, the cells on the samples were stained with fluorescent phalloidin for 20 min and with DAPI (4′, 6-diamidino-2-phenylindole) for 5 min before the observation of cell morphology on the sample surface. The level of cell proliferation was measured using metoxyphenyl tetrazolium salt (MTS) assay (CellTiter 96 Aqueous One Solution, Promega, USA) after 3 and 5 days of culturing of cells on the surfaces of the hydrogels. 2.5. In Vivo Evaluation of Hydrogels. Pure HAc and pHAc-30 wt % CaP hydrogels were implanted in intramuscular sites of 6-week old Sprague−Dawley (SD) rats. The dorsal skin of the rats was incised using a surgical blade, and the hydrogels were implanted with a volume of 0.4 mL. The rats were sacrificed 4 and 8 weeks after implantation. The tissues extracted with the hydrogel samples were immediately fixed in 10% formalin, gradually dehydrated with ethanol, and embedded in paraffin for histological analysis. The prepared paraffin blocks were sectioned at a thickness of 4 μm in the longitudinal axis of the hydrogels in the blocks, which was perpendicular to the skin surface. The slices were stained with Alcian blue to detect the remnants of the HAc-based hydrogels in the implanted region. In vivo stability of the hydrogels was evaluated by observing the shape and amount of the remaining hydrogels. In particular, the remaining amount was estimated by normalizing the cross-sectional area of the hydrogels using the computerized image analysis system, SPOT (Diagnostic Instrument, Sterling Heights, MI). The in vivo biocompatibility of the hydrogels was also assessed histologically after staining with Masson’s trichrome (M&T). All in vivo animal experiments were performed according to the protocol (no. 15-0075) approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. The use and handling of the animals in the study was abide by the Guide for the Care and Use of Laboratory Animals of Seoul National University. 2.6. Statistical Analysis. All experimental results were expressed as the mean ± standard deviation (SD) for n > 3. The difference between the two groups was determined using a one-way analysis of variance (ANOVA) and p < 0.05 and p < 0.01 were considered to be statistically significant (*p < 0.05 and **p < 0.01).
Figure 1. Cross-sectional morphology of (A) pure HAc and (B) pHAc-30 wt % CaP hydrogels and surface morphology of (C) pure HAc and (D) pHAc-30 wt % CaP hydrogels in higher magnitude (× 30 K).
Information). It is a noteworthy fact that only particle size is varied with the CaP content of the pHAc-CaP hydrogels. Therefore, the CaP content of pHAc-CaP hydrogels can be modulated by tuning the size of CaP nanoparticles. The nature of CaP precipitates within the pHAc-CaP hydrogels was further investigated by TEM, as shown in Figure 2. TEM micrographs showed the presence of identical primary nanoparticles with highly spherical shapes and a uniform size of 200 nm. Apparently, no agglomeration occurred, leaving a large surface area available for interaction with polymer chains. For the phase detection, the selected-area electron diffraction (SAED) pattern of the CaP nanoparticles was observed. The SAED pattern indicated a mainly amorphous state of the CaP nanoparticles because the diffraction contrast for these spherical particles was weak. EDS mapping confirmed that these nanoparticles were composed of calcium, phosphorus, and oxygen. Moreover, the quantitative EDS analyses of these nanoparticles indicated a composition of 18 atomic% Ca−17 atomic% P−65 atomic% O, implying the expected Ca/P ratio of the CaP precipitates was ∼1.05. The composition and phase of the nanoparticles were also identified through XRD analysis, as shown in Figure 3. The XRD pattern of the pure HAc hydrogel showed no significant peaks, as expected (Figure 3A). The XRD of the nanocomposite was slightly different from that of pure HAc. A broad peak at 32−35° indicated that the calcium phosphate precipitates had low crystallinity (Figure 3B). Under mild conditions with low temperature, amorphous CaP particles were precipitated. When the composite was heat-treated at 1100 °C in air, two peaks were detected as shown in Figure 3C. These peaks were identified to be calcium phosphate phases, Ca2P2O7 and Ca3(PO2)2, indicating a Ca/P ratio of 1 to 1.5. On the basis of these observations, it is easily inferred that the nanoparticles were not freely precipitated in the solution. Instead HAc played an important role in forming small and uniform CaP particles. One of the key parameters is the negatively charged carboxyl groups in HAc. The positively charged calcium ions are supposed to be attracted to reaction sites containing carboxyl groups. Subsequent reactions to form CaP nanoparticles should then occur at those sites, leading to homogeneous distribution of the particles. Excessive growth of
3. RESULTS AND DISCUSSION 3.1. Characterization of pHAc-CaP Hydrogels. The microstructures of the hydrogels are shown in Figure 1. Pure HAc hydrogels typically have porous microstructures with pore sizes of 200−500 μm, as shown in Figure 1A. The pHAc-CaP hydrogels with 30 wt % CaP also showed a highly porous structure with interconnected pores, implying that the network structure of the hydrogel is maintained after the in situ process (Figure 1B); however, at higher magnification, significant differences in the surface morphologies of pure HAc and pHAc-CaP hydrogels emerged. A smooth surface matrix was observed on the pure HAc hydrogel, as indicated in Figure 1C, while a nanoroughened surface with 200−350 nm spherical nanoparticles was observed on hydrogels with 30 wt % CaP (Figure 1D). Like pHAc-30 wt % CaP hydrogel, the pHAc-CaP hydrogels with different CaP contents showed dense distribution of uniform-size spherical CaP nanoparticles through HAc matrix (images shown in the Supporting C
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 2. Typical TEM image of CaP nanoparticles in the pHAc-CaP hydrogels with EDS mapping analysis (inset: the select-area electron diffraction (SAED) patterns of CaP nanoparticles).
3.2. Mechanical Properties of pHAc-CaP Hydrogels. The effects of CaP content on the mechanical properties of pHAc-CaP hydrogels were explored using a strain-controlled rheometer as shown in Figure 4. The storage modulus (G′) of pHAc-CaP was significantly higher than that of pure HAc hydrogel. As the CaP content increased up to 40 wt %, G′ increased steadily, as shown in Figure 4A. G′ of pHAc-CaP exhibited the frequencyindependent behavior, whereas G′ of pure HAc changed over frequency. That means that the elasticity of the HAc hydrogel was markedly enhanced by incorporating the CaP nanoparticles through the in situ process. Complex viscosity measurements were also performed as shown in Figure 4B. All the hydrogels showed shear thinning phenomena under deformation. The complex viscosity of pHAc-CaP hydrogels was considerably higher at all shear rates, compared with that of pure HAc hydrogels. Furthermore, the concentration of CaP had a positive influence on the viscosity at low frequencies. These results implied that CaP nanoparticles became strongly integrated with the HAc matrix after in situ precipitation. To elucidate the role of precipitated CaP nanoparticles, the viscoelastic behavior of the hydrogel systems was observed by
Figure 3. XRD patterns of (a) as-prepared pure HAc hydrogel, (b) asprepared pHAc-CaP hydrogel, and (c) pHAc-CaP hydrogel after heat treatment at 1100 °C.
the particles was also effectively suppressed by the polymeric chains.23,27,35−37
Figure 4. Rheological behavior of pHAc-CaP hydrogels: (A) storage modulus over frequency with different CaP amount and (B) complex viscosity as a function of frequency. D
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 5. (A) Storage moduli of pure HAc, pHAc-30 wt % CaP, and mHAc-30 wt % CaP hydrogels as a function of frequency. The experimental results (experiment) of storage moduli were compared with model predictions (curve fitting). (B) Storage moduli of pure HAc, pHAc-30 wt % CaP, and mHAc-30 wt % CaP hydrogels as a function of strain.
Figure 6. Schematic (left) and surface morphology (right) of (A) pHAc-CaP-30 wt % and (B) mHAc-CaP-30 wt %.
structural morphology on a microscopic scale.38−42 On the basis of the theory of the absorption energy between polymer chains and nanoparticles, a frequency-dependent modulus,G′comp (ϖ), was calculated according to the fraction of CaP adsorption to the HAc matrix, as follows
both frequency sweep and strain sweep, and the results were compared with those of the mHAc-CaP system (Figure 5). The storage modulus (G′) of pure HAc hydrogel showed frequency-dependent elastic behavior, whereas the G′ of the nanocomposites was relatively consistent. More interestingly, the storage moduli of pHAc-CaP were consistently about five times larger than those of mHAc-CaP. At this point, it is noteworthy to describe a theoretical model explaining viscoelastic behaviors on a macroscopic scale in relation to
′ (ϖ) = νCPCSGCPCS′(ϖ) + νFCSG FCS′(ϖ) Gcomp
where νCPCS and νFCS represent the volume fractions of CaP absorbed chains and free chains, respectively. GFCS′(ϖ) and E
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
shown in Figure 6A, which is clearly different from the mHAcCaP hydrogels, which have a lower density of attached chains (Figure 6B). These pHAc-CaP hydrogel networks are believed to originate from the CaP nanoparticles acting as physical crosslinkers.45−48 As mentioned in the previous section, the chelating effect of ionized carboxyl groups of HAc and calcium ions during in situ CaP formation is believed to promote apatite nucleation through electrostatic interactions.49−51 During immersion of the HAc hydrogels in the solution of CaCl2 and H3PO4, the carboxyl groups of HAc chains likely exist in a neutral form, with calcium and phosphate ions uniformly distributed in the HAc network. By increasing the pH of the solution to 11, solubility of calcium and phosphate ions is rapidly lowered, and CaP precipitates start being formed. Meanwhile, the calcium ions are supposed to be chelated with the ionized carboxyl groups of HAc at higher pH. Therefore, the nucleation and precipitation of CaP are initiated from the surface of HAc hydrogels.35,36 The surface morphologies of the specimens also support the suggested structural model with regard to the molecular interaction mechanisms between HAc chains and CaP nanoparticles (Figure 6 (right)). The surface morphology of the pHAc-CaP exhibited homogeneously dispersed CaP nanoparticles on the HAc surface (Figure 6A). During the in situ precipitation, those nanoparticles were uniformly formed on HAc with minimal agglomeration. On the contrary, the surface morphology of the mHAc-CaP was inhomogeneous due to the nonuniform distribution of agglomerated CaP particles (Figure 6B). The particles were observed to form clusters through particle agglomeration during physical mixing. 3.3. In Vitro Biostability of Hydrogels. The swelling behavior of the pure HAc, pHAc-CaP, and mHAc-CaP hydrogels was investigated, as shown in Figure 7. The pure HAc hydrogel had an equilibrium swelling ratio of 110. The swelling ratios of the nanocomposite hydrogels were reduced as the CaP content increased; however, the amount of water influx in the pHAc-CaP hydrogels was significantly lower than in the mHAc-CaP hydrogels. The dramatic reduction in the swelling of pHAc-CaP was attributed mainly to the increased overall cross-link density of the hydrogel network.52
Figure 7. Swelling ratios of the hydrogels after 24 h immersed in PBS at 37 °C.
GCPCS′(ϖ) stand for the stiffness of the CaP absorbed chain segment and the free chain segment, respectively. (See the Supporting Information for further details.) GFCS′(ϖ) was obtained from the pure HAc data and GCPCS′(ϖ) was approximated through the energy affinity from adsorbed chains and CaP particles.39,43,44 The numerical curve fitting followed the trends of the viscoelastic response in the experimental results, as shown in Figure 5A. In addition, significant enhancement on mechanical stability of pHAc-CaP was observed from the strain sweep results, compared with both mHAc-CaP and pure HAc hydrogels (Figure 5B). pHAcCaP exhibited noticeably larger linear viscoelastic region than mHAc-CaP, maintaining its structure integrity until 20% of strain, whereas mHAc-CaP showed network breakdown from 7% of strain with decrease in storage modulus. The enhanced elastic reinforcement and mechanical stability of pHAc-CaP indicated stronger binding between HAc chains and CaP particles. On the contrary, the low storage modulus and high nonlinearity of mHAc-CaP can be attributed to the weak polymer−particle interaction and strong aggregation of CaP particles. In accordance with the results, the overall schematic model of the composite hydrogels is depicted in Figure 6 (left). The pHAc-CaP hydrogels are deemed to have a greater proportion of adsorbed HAc chains around CaP particles, as
Figure 8. In vitro enzymatic degradation behavior of pure HAc, mHAc-CaP, and pHAc-CaP hydrogels: (A) Comparison on degradation rates as a function of incubated time (B) remaining weight after 4 h of degradation with CaP content. F
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 9. In vitro cellular response of pure HAc and pHAc-CaP hydrogels: (A) typical CLSM images of L929 fibroblasts cultured on pure HAc and pHAc-CaP hydrogels for 3 days and (B) cell viability of pure HAc and pHAc-CaP hydrogels measured by MTS assay after 3 and 5 days.
the weight change as a function of incubation time as shown in Figure 8A. The pure HAc hydrogels were degraded most rapidly as expected. The pHAc-CaP hydrogels had improved resistance to enzymatic degradation compared with pure HAc and mHAcCaP with the same CaP content. After 2 days, 40 wt % of pHAc-CaP still remained, maintaining the shape of the hydrogel, whereas both pure HAc and mHAc-CaP were completely degraded. The optical images of the remnant hydrogels after degradation clearly showed that the pHAc-CaP hydrogels had superior biostability. This enhanced resistance to enzymatic degradation was attributed to the CaP nanoparticles in pHAc-CaP hydrogels, which effectively block the access of the hyaluronidase by means of steric hindrance.53,54 The degradation ratio for pHAc-CaP, depending on the CaP content, was also compared by calculating the relative remaining weight after 4 h of enzymatic degradation (Figure 8B). In the hyaluronidase solution, pHAc-CaP hydrogels underwent less hydrolytic degradation, as increasing CaP content led to less swelling. 3.4. Biological Performances of Hydrogels. From the mechanical and in vitro biostability tests, the advantages of pHAc-CaP over mHAc-CaP were clearly confirmed, implying that pHAc-CaP has great potential as a biomaterial for tissue engineering. In this context, in terms of biological performance, particularly, biostability and bioactivity, the pHAc-CaP systems were carefully evaluated through in vitro and in vivo tests, compared with pure HAc that is the well-known and widely used system as a reference. Above all, to evaluate cytocompatibility of pHAc-CaP nanocomposite hydrogels, the cell proliferation tests were carried out. The in vitro cellular responses of fibroblast cells on the hydrogels are shown in Figure 9. All cells on pHAc-CaP hydrogels grew well with no sign of cytotoxicity. The morphology of fibroblasts on the pHAc-CaP hydrogels appeared more polarized and stretched in contrast with the round-shaped cells on the pure HAc hydrogels (Figure 9A). The cell spreading area of nanocomposite hydrogels (∼400 μm2) was significantly larger than that of pure HAc (∼250 μm2). (See Supplemental Figure S2 for further details.) The values of cell spreading area among pHAc-CaP nanocomposites were comparable, showing no significant difference. Also, the cell densities on the pHAc-CaP hydrogel surfaces were apparently higher than that on the pure HAc hydrogels. This
Figure 10. In vivo enzymatic degradation behavior of pure HAc and pHAc-CaP hydrogels: (A) histological images of pure HAc (top) and mHAc-30 wt % CaP hydrogel (bottom) stained by Alcian blue after 4 weeks of implantation and (B) remaining area ratio of pure HAc and pHAc-30 wt % CaP hydrogels after 4 and 8 weeks of implantation.
The in vitro biodegradability of the hydrogels was assessed by immersing them in hyaluronidase solution and monitoring G
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 11. In vivo bioactivity test of hydrogels injection on subcutaneous tissue of SD rat: histological images (left) of (A) pure HAc and (B) pHAc30 wt % CaP hydrogel stained by M&T after 4 weeks of implantation and higher magnitude images (right) of the histological images.
degraded (10% remnant), in contrast with ∼50% remaining for the pHAc-CaP hydrogel. The bioactivity of the hydrogel was evaluated by histological analyses. Collagen formation in the pHAc-CaP hydrogels observed by M&T staining was significantly greater than that of the pure HAc hydrogels, as shown in Figure 11, indicating that CaP nanoparticles provide a highly effective microenvironment for fibroblast cell proliferation and ECM production.61 These results clearly indicated that CaP nanoparticles not only improve the dimensional stability of pHAc-CaP hydrogels over a period of time by hindering enzymatic degradation but also stimulate the cellular environments to generate collagen, which can enhance the structural support of the hydrogels and the surrounding ECM.57,64 More specifically, stimulated by exposed CaP nanoparticles on the surface of pHAc-CaP hydrogels, fibroblast cells from surrounding tissues migrated to the area around pHAc-CaP hydrogels and regenerated a new tissue structure, including collagen deposition and vascularization.64,65 Thus, through the in situ precipitation process, HAcCaP nanocomposite with long-term biostability and excellent bioactivity was generated. The produced hydrogels have great potential for soft tissue engineering applications.
observation showed good agreement with the quantified cell density determined through the MTS assay, as shown in Figure 9B. The level of fibroblast proliferation on the pHAc-CaP hydrogels after 5 days of culture was about 8 times higher than that on the pure HAc hydrogels, implying enhanced biocompatibility for the nanocomposite hydrogels. In most cases, phosphate-containing biomaterial scaffolds have positive effects in promoting cellular response.55,56 In the present system, the nanosized adhesive sites of CaP nanoparticles in pHAc-CaP hydrogels might have provided physical attachment for the fibroblast cells.57 In addition, the mechanical stability of the hydrogels is believed to have positive effects on fibroblast behavior.58−60 Interestingly, no significant difference in cell proliferation levels was observed among the pHAc-CaP composite hydrogels with different CaP content. The in vivo biostability of HAc hydrogels is one of the major obstacles for suitable resistance to degradation, which is a prerequisite for all biomedical scaffold applications.40,61−63 It is well known that HAc hydrogel itself has limited biostability due to its inherent structural instability as well as enzymatic reactions in the body. We investigated the in vivo stability and bioactivity of pHAc-CaP hydrogels through volume retention and histological analysis, respectively, after 4 and 8 weeks of implantation in subcutaneous sites in rats. The histological images of the hydrogel-tissue paraffin blocks were used to determine the amount of remaining hydrogels. As shown in Figure 10A, the thickness and diameter of the cross-sectioned pure HAc hydrogel decreased markedly, indicating that a large portion of the hydrogel was degraded in the body, even after 4 weeks of implantation. On the contrary, the pHAc-30 wt % CaP was found to maintain its original shape with good structural integrity after 4 and 8 weeks of implantation. From image analysis, the remaining area was quantified as shown in Figure 10B. After the 4 weeks of implantation, only 20% of the pure HAc hydrogels remained in the body, whereas >50% of pHAc-30 wt % CaP remained. After 8 weeks, the pure hydrogel was almost
5. CONCLUSIONS By introducing in situ precipitation within a HAc hydrogel matrix, we developed HAc-CaP nanocomposite hydrogels with significantly enhanced mechanical strength as well as degradation resistance. The resulting structural homogeneity, integrity, and minimal nanoparticle aggregation yield positive effects on the mechanical properties and degradation kinetics compared with the microscopic structure obtained from a physical mixing process. pHAc-CaP hydrogels were successfully fabricated, with uniformly precipitated CaP nanoparticles strongly conjugated with HAc polymer chains. With structurally homogeneous architecture, enhanced physical cross-links, and a high density of entanglements by precipitated CaP nanoparticles, pHAc-CaP hydrogels exhibited significantly reduced H
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
human mesenchymal stem cells. Biomaterials 2007, 28 (10), 1830− 1837. (9) Tan, H. P.; Marra, K. G. Injectable, Biodegradable Hydrogels for Tissue Engineering Applications. Materials 2010, 3 (3), 1746−1767. (10) Yeom, J.; Bhang, S. H.; Kim, B. S.; Seo, M. S.; Hwang, E. J.; Cho, I. H.; Park, J. K.; Hahn, S. K. Effect of Cross-Linking Reagents for Hyaluronic Acid Hydrogel Dermal Fillers on Tissue Augmentation and Regeneration. Bioconjugate Chem. 2010, 21 (2), 240−247. (11) Xu, X.; Jha, A. K.; Harrington, D. A.; Farach-Carson, M. C.; Jia, X. Q. Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter 2012, 8 (12), 3280−3294. (12) Drury, J. L.; Mooney, D. J. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 2003, 24 (24), 4337−4351. (13) Jia, X. Q.; Kiick, K. L. Hybrid Multicomponent Hydrogels for Tissue Engineering. Macromol. Biosci. 2009, 9 (2), 140−156. (14) Segura, T.; Anderson, B. C.; Chung, P. H.; Webber, R. E.; Shull, K. R.; Shea, L. D. Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern. Biomaterials 2005, 26 (4), 359−371. (15) Pitarresi, G.; Pierro, P.; Palumbo, F. S.; Tripodo, G.; Giammona, G. Photo-cross-linked hydrogels with polysaccharide-poly(amino acid) structure: New biomaterials for pharmaceutical applications. Biomacromolecules 2006, 7 (4), 1302−1310. (16) Jia, X. Q.; Burdick, J. A.; Kobler, J.; Clifton, R. J.; Rosowski, J. J.; Zeitels, S. M.; Langer, R. Synthesis and characterization of in situ cross-linkable hyaluronic acid-based hydrogels with potential application for vocal fold regeneration. Macromolecules 2004, 37 (9), 3239− 3248. (17) Hahn, S. K.; Ohri, R.; Giachelli, C. M. Anti-calcification of bovine pericardium for bioprosthetic heart valves after surface modification with hyaluronic acid derivatives. Biotechnol. Bioprocess Eng. 2005, 10 (3), 218−224. (18) Dickerson, M. B.; Sandhage, K. H.; Naik, R. R. Protein- and Peptide-Directed Syntheses of Inorganic Materials. Chem. Rev. 2008, 108 (11), 4935−4978. (19) Sohrabi, M.; Hesaraki, S.; Kazemzadeh, A.; Alizadeh, M. Development of injectable biocomposites from hyaluronic acid and bioactive glass nano-particles obtained from different sol-gel routes. Mater. Sci. Eng., C 2013, 33 (7), 3730−3744. (20) Pek, Y. S.; Kurisawa, M.; Gao, S.; Chung, J. E.; Ying, J. Y. The development of a nanocrystalline apatite reinforced crosslinked hyaluronic acid-tyramine composite as an injectable bone cement. Biomaterials 2009, 30 (5), 822−828. (21) Lau, H. K.; Kiick, K. L. Opportunities for Multicomponent Hybrid Hydrogels in Biomedical Applications. Biomacromolecules 2015, 16 (1), 28−42. (22) Gaharwar, A. K.; Schexnailder, P. J.; Jin, Q.; Wu, C. J.; Schmidt, G. Addition of Chitosan to Silicate Cross-Linked PEO for Tuning Osteoblast Cell Adhesion and Mineralization. ACS Appl. Mater. Interfaces 2010, 2 (11), 3119−3127. (23) Gaharwar, A. K.; Dammu, S. A.; Canter, J. M.; Wu, C. J.; Schmidt, G. Highly Extensible, Tough, and Elastomeric Nanocomposite Hydrogels from Poly(ethylene glycol) and Hydroxyapatite Nanoparticles. Biomacromolecules 2011, 12 (5), 1641−1650. (24) Valles-Lluch, A.; Poveda-Reyes, S.; Amoros, P.; Beltran, D.; Pradas, M. M. Hyaluronic Acid-Silica Nanohybrid Gels. Biomacromolecules 2013, 14 (12), 4217−4225. (25) Nejadnik, M. R.; Yang, X.; Bongio, M.; Alghamdi, H. S.; Van den Beucken, J. J. J. P.; Huysmans, M. C.; Jansen, J. A.; Hilborn, J.; Ossipov, D.; Leeuwenburgh, S. C. G. Self-healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and calcium phosphate nanoparticles. Biomaterials 2014, 35 (25), 6918−6929. (26) Yokoi, T.; Kawashita, M.; Kikuta, K.; Ohtsuki, C. Biomimetic mineralization of calcium phosphate crystals in polyacrylamide hydrogel: Effect of concentrations of calcium and phosphate ions on crystalline phases and morphology. Mater. Sci. Eng., C 2010, 30 (1), 154−159. (27) Li, Z. Y.; Su, Y. L.; Xie, B. Q.; Wang, H. L.; Wen, T.; He, C. C.; Shen, H.; Wu, D. C.; Wang, D. J. A tough hydrogel-hydroxyapatite
in vitro and in vivo degradation rates and dramatic improvement of the mechanical moduli with highly elastic behavior. The biocompatibility of the composite hydrogels was clearly enhanced compared with that of pure HAc and mHAc-CaP hydrogels. They therefore exhibit significant potential for various biomedical applications.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01557. Theoretical and empirical calcium phosphate (CaP) content of pHAc-CaP hydrogels; water content of fully swollen HAc and HAc-CaP nanocomposite hydrogels; surface morphology of various pHAc-CaP hydrogels; modeling details of the viscoelastic behavior of pHAcCaP and mHAc-CaP hydrogels; and quantified cell spreading area of fibroblasts on pure HAc and pHAc-CaP hydrogels. (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +82 2 880 8320. Fax: +82 2 884 1413. E-mail: sat105@ snu.ac.kr. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research was supported by the R&D Program (S2174750) funded by the Small and Medium Business Administration (SMBA, Korea).
■
REFERENCES
(1) Bencherif, S. A.; Srinivasan, A.; Horkay, F.; Hollinger, J. O.; Matyjaszewski, K.; Washburn, N. R. Influence of the degree of methacrylation on hyaluronic acid hydrogels properties. Biomaterials 2008, 29 (12), 1739−1749. (2) Hachet, E.; Van Den Berghe, H.; Bayma, E.; Block, M. R.; AuzelyVelty, R. Design of Biomimetic Cell-Interactive Substrates Using Hyaluronic Acid Hydrogels with Tunable Mechanical Properties. Biomacromolecules 2012, 13 (6), 1818−1827. (3) Burdick, J. A.; Prestwich, G. D. Hyaluronic Acid Hydrogels for Biomedical Applications. Adv. Mater. 2011, 23 (12), H41−H56. (4) Widjaja, L. K.; Bora, M.; Chan, P. N. P. H.; Lipik, V.; Wong, T. T. L.; Venkatraman, S. S. Hyaluronic acid-based nanocomposite hydrogels for ocular drug delivery applications. J. Biomed. Mater. Res., Part A 2014, 102 (9), 3056−3065. (5) Schramm, C.; Spitzer, M. S.; Henke-Fahle, S.; Steinmetz, G.; Januschowski, K.; Heiduschka, P.; Geis-Gerstorfer, J.; Biedermann, T.; Bartz-Schmidt, K. U.; Szurman, P. The Cross-linked Biopolymer Hyaluronic Acid as an Artificial Vitreous Substitute. Invest. Ophthalmol. Visual Sci. 2012, 53 (2), 613−621. (6) Kurisawa, M.; Chung, J. E.; Yang, Y. Y.; Gao, S. J.; Uyama, H. Injectable biodegradable hydrogels composed of hyaluronic acidtyramine conjugates for drug delivery and tissue engineering. Chem. Commun. 2005, 34, 4312−4314. (7) Yu, F.; Cao, X. D.; Li, Y. L.; Zeng, L.; Yuan, B.; Chen, X. F. An injectable hyaluronic acid/PEG hydrogel for cartilage tissue engineering formed by integrating enzymatic crosslinking and Diels-Alder ″click chemistry″. Polym. Chem. 2014, 5 (3), 1082−1090. (8) Kim, J.; Kim, I. S.; Cho, T. H.; Lee, K. B.; Hwang, S. J.; Tae, G.; Noh, I.; Lee, S. H.; Park, Y.; Sun, K. Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and I
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules bone-like composite fabricated in situ by the electrophoresis approach. J. Mater. Chem. B 2013, 1 (12), 1755−1764. (28) Xianmiao, C.; Yubao, L.; Yi, Z.; Li, Z.; Jidong, L.; Huanan, W. Properties and in vitro biological evaluation of nano-hydroxyapatite/ chitosan membranes for bone guided regeneration. Mater. Sci. Eng., C 2009, 29 (1), 29−35. (29) Parhi, P.; Ramanan, A.; Ray, A. R. Preparation and characterization of alginate and hydroxyapatite-based biocomposite. J. Appl. Polym. Sci. 2006, 102 (6), 5162−5165. (30) Yamaguchi, I.; Tokuchi, K.; Fukuzaki, H.; Koyama, Y.; Takakuda, K.; Monma, H.; Tanaka, T. Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites. J. Biomed. Mater. Res. 2001, 55 (1), 20−27. (31) Wang, L.; Li, Y.; Li, C. Z. In situ processing and properties of nanostructured hydroxyapatite/alginate composite. J. Nanopart. Res. 2009, 11 (3), 691−699. (32) He, L. H.; Yao, L.; Xue, R.; Sun, J.; Song, R. In-situ mineralization of chitosan/calcium phosphate composite and the effect of solvent on the structure. Front Mater. Sci. 2011, 5 (3), 282− 292. (33) Nardecchia, S.; Gutierrez, M. C.; Serrano, M. C.; Dentini, M.; Barbetta, A.; Ferrer, M. L.; del Monte, F. In Situ Precipitation of Amorphous Calcium Phosphate and Ciprofloxacin Crystals during the Formation of Chitosan Hydrogels and Its Application for Drug Delivery Purposes. Langmuir 2012, 28 (45), 15937−15946. (34) Leach, J. B.; Bivens, K. A.; Patrick, C. W., Jr.; Schmidt, C. E. Photocrosslinked hyaluronic acid hydrogels: Natural, biodegradable tissue engineering scaffolds. Biotechnol. Bioeng. 2003, 82 (5), 578−589. (35) Jiao, Y.; Gyawali, D.; Stark, J. M.; Akcora, P.; Nair, P.; Tran, R. T.; Yang, J. A rheological study of biodegradable injectable PEGMC/ HA composite scaffolds. Soft Matter 2012, 8 (5), 1499−1507. (36) Li, Q. H.; Li, M.; Zhu, P. Z.; Wei, S. C. In vitro synthesis of bioactive hydroxyapatite using sodium hyaluronate as a template. J. Mater. Chem. 2012, 22 (38), 20257−20265. (37) Li, B. Q.; Wang, Y. L.; Jia, D. C.; Zhou, Y. Gradient Structural Bone-Like Apatite Induced by Chitosan Hydrogel via Ion Assembly. J. Biomater. Sci., Polym. Ed. 2011, 22 (4−6), 505−517. (38) Shibayama, M. Structure-mechanical property relationship of tough hydrogels. Soft Matter 2012, 8 (31), 8030−8038. (39) Lee, S.; Tirumala, V. R.; Nagao, M.; Tominaga, T.; Lin, E. K.; Gong, J. P.; Wu, W. L. Dynamics in Multicomponent Polyelectrolyte Solutions. Macromolecules 2009, 42 (4), 1293−1299. (40) Sarvestani, A. S.; Jabbari, E. A model for the viscoelastic Behavior of nanofilled hydrogel composites under oscillatory shear loading. Polym. Compos. 2008, 29 (3), 326−336. (41) Merabia, S.; Sotta, P.; Long, D. R. Unique Plastic and Recovery Behavior of Nanofilled Elastomers and Thermoplastic Elastomers (Payne and Mullins Effects). J. Polym. Sci., Part B: Polym. Phys. 2010, 48 (13), 1495−1508. (42) Sarvestani, A. S.; He, X. Z.; Jabbari, E. The role of filler-matrix interaction on viscoelastic response of biomimetic nanocomposite hydrogels. J. Nanomater. 2008, 2008, 1. (43) Degennes, P. G. Polymers at an Interface - a Simplified View. Adv. Colloid Interface Sci. 1987, 27 (3−4), 189−209. (44) Degennes, P. G. Polymers at an Interface 0.2. Interaction between 2 Plates Carrying Adsorbed Polymer Layers. Macromolecules 1982, 15 (2), 492−500. (45) Carlsson, L.; Rose, S.; Hourdet, D.; Marcellan, A. Nano-hybrid self-crosslinked PDMA/silica hydrogels. Soft Matter 2010, 6 (15), 3619−3631. (46) Wang, X. R.; Robertson, C. G. Strain-induced nonlinearity of filled rubbers. Phys. Rev. E 2005, 72 (3), 031406. (47) Wang, M.; Deb, S.; Bonfield, W. Chemically coupled hydroxyapatite-polyethylene composites: processing and characterisation. Mater. Lett. 2000, 44 (2), 119−124. (48) Sarvestani, A. S.; He, X. Z.; Jabbari, E. Effect of osteonectinderived peptide on the viscoelasticity of hydrogel/apatite nanocomposite scaffolds. Biopolymers 2007, 85 (4), 370−378.
(49) Oyane, A.; Uchida, M.; Yokoyama, Y.; Choong, C.; Triffitt, J.; Ito, A. Simple surface modification of poly(epsilon-caprolactone) to induce its apatite-forming ability. J. Biomed. Mater. Res., Part A 2005, 75A (1), 138−145. (50) Oyane, A.; Uchida, M.; Choong, C.; Triffitt, J.; Jones, J.; Ito, A. Simple surface modification of poly(epsilon-caprolactone) for apatite deposition from simulated body fluid. Biomaterials 2005, 26 (15), 2407−2413. (51) Goes, J. C.; Figueiro, S. D.; Oliveira, A. M.; Macedo, A. A. M.; Silva, C. C.; Ricardo, N. M. P. S.; Sombra, A. S. B. Apatite coating on anionic and native collagen films by an alternate soaking process. Acta Biomater. 2007, 3 (5), 773−778. (52) Gaharwar, A. K.; Schexnailder, P. J.; Kline, B. P.; Schmidt, G. Assessment of using Laponite (R) cross-linked poly(ethylene oxide) for controlled cell adhesion and mineralization. Acta Biomater. 2011, 7 (2), 568−577. (53) Desimone, M. F.; Helary, C.; Quignard, S.; Rietveld, I. B.; Bataille, I.; Copello, G. J.; Mosser, G.; Giraud-Guille, M. M.; Livage, J.; Meddahi-Pelle, A.; Coradin, T. In vitro studies and preliminary in vivo evaluation of silicified concentrated collagen hydrogels. ACS Appl. Mater. Interfaces 2011, 3 (10), 3831−8. (54) Nejadnik, M. R.; Yang, X.; Bongio, M.; Alghamdi, H. S.; van den Beucken, J. J.; Huysmans, M. C.; Jansen, J. A.; Hilborn, J.; Ossipov, D.; Leeuwenburgh, S. C. Self-healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and calcium phosphate nanoparticles. Biomaterials 2014, 35 (25), 6918−29. (55) Murphy, W. L.; Mooney, D. J. Bioinspired growth of crystalline carbonate apatite on biodegradable polymer substrata. J. Am. Chem. Soc. 2002, 124 (9), 1910−1917. (56) Nuttelman, C. R.; Tripodi, M. C.; Anseth, K. S. Synthetic hydrogel niches that promote hMSC viability. Matrix Biol. 2005, 24 (3), 208−218. (57) Bongio, M.; van den Beucken, J. J.; Nejadnik, M. R.; Leeuwenburgh, S. C.; Kinard, L. A.; Kasper, F. K.; Mikos, A. G.; Jansen, J. A. Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior. Eur. Cells Mater. 2011, 22, 359−76. (58) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 2006, 126 (4), 677−689. (59) Discher, D. E.; Janmey, P.; Wang, Y. L. Tissue cells feel and respond to the stiffness of their substrate. Science 2005, 310 (5751), 1139−1143. (60) Wang, L. S.; Chung, J. E.; Chan, P. P. Y.; Kurisawa, M. Injectable biodegradable hydrogels with tunable mechanical properties for the stimulation of neurogenesic differentiation of human mesenchymal stem cells in 3D culture. Biomaterials 2010, 31 (6), 1148−1157. (61) Hwang, C. M.; Ay, B.; Kaplan, D. L.; Rubin, J. P.; Marra, K. G.; Atala, A.; Yoo, J. J.; Lee, S. J. Assessments of injectable alginate particle-embedded fibrin hydrogels for soft tissue reconstruction. Biomed. Mater. 2013, 8 (1), 014105. (62) Largo, R. A.; Ramakrishnan, V. M.; Marschall, J. S.; Ziogas, A.; Banfi, A.; Eberli, D.; Ehrbar, M. Long-term biostability and bioactivity of ″fibrin linked″ VEGF(121) in vitro and in vivo. Biomater. Sci. 2014, 2 (4), 581−590. (63) Stosich, M. S.; Moioli, E. K.; Wu, J. K.; Lee, C. H.; Rohde, C.; Yoursef, A. M.; Ascherman, J.; Diraddo, R.; Marion, N. W.; Mao, J. J. Bioengineering strategies to generate vascularized soft tissue grafts with sustained shape. Methods 2009, 47 (2), 116−121. (64) Quan, T. H.; Wang, F.; Shao, Y.; Rittie, L.; Xia, W.; Orringer, J. S.; Voorhees, J. J.; Fisher, G. J. Enhancing Structural Support of the Dermal Microenvironment Activates Fibroblasts, Endothelial Cells, and Keratinocytes in Aged Human Skin In Vivo. J. Invest. Dermatol. 2013, 133 (3), 658−667. (65) Kim, Z. H.; Lee, Y.; Kim, S. M.; Kim, H.; Yun, C. K.; Choi, Y. S. A Composite Dermal Filler Comprising Cross-Linked Hyaluronic Acid J
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules and Human Collagen for Tissue Reconstruction. J. Microbiol. Biotechnol. 2015, 25 (3), 399−406.
K
DOI: 10.1021/acs.biomac.5b01557 Biomacromolecules XXXX, XXX, XXX−XXX
