Hyaluronic acid and fibrin from L-PRP form semi-IPNs with tunable properties suitable for use in regenerative medicine

Materials Science & Engineering C 109 (2020) 110547

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Hyaluronic acid and fibrin from L-PRP form semi-IPNs with tunable properties suitable for use in regenerative medicine

T

Bruna Alice Gomes de Meloa, Carla Giometti Françaa, José Luis Dávilab, Nilza Alzira Batistac, Carolina Caliari-Oliveirad, Marcos Akira d'Ávilab, Ângela Cristina Malheiros Luzoe, ⁎ José Fabio Santos Duarte Lanaf, Maria Helena Andrade Santanaa, a

Department of Engineering of Materials and Bioprocesses, School of Chemical Engineering, University of Campinas, 13083-852 Campinas, SP, Brazil Department of Manufacturing and Materials Engineering, School of Mechanical Engineering, University of Campinas, 13083-860 Campinas, SP, Brazil c Orthopaedic Biomaterials Laboratory, Faculty of Medical Sciences, University of Campinas, 13083-887 Campinas, SP, Brazil d In Situ Cell Therapy, Supera Innovation and Technology Park, 14056-680 Ribeirão Preto, SP, Brazil e Haematology & Hemotherapy Center, Umbilical Cord Blood Bank, University of Campinas, 13083-878 Campinas, SP, Brazil f Bone and Cartilage Institute, 13334-170 Indaiatuba, SP, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Platelet Leukocyte Fibrin Hyaluronic acid Mesenchymal stem cell

Autologous leukocyte- and platelet-rich plasma (L-PRP) combined with hyaluronic acid (HA) has been widely used in local applications for cartilage and bone regeneration. The association between L-PRP and HA confers structural and rheological changes that differ among individual biomaterials but has not been investigated. Therefore, the standardization and characterization of L-PRP-HA are important to consider when comparing performance results to improve future clinical applications. To this end, we prepared semi-interpenetrating polymer networks (semi-IPNs) of L-PRP and HA and characterized their polymerization kinetics, morphology, swelling ratio, stability and rheological behavior, which we found to be tunable according to the HA molar mass (MM). Mesenchymal stem cells derived from human adipose tissue (h-AdMSCs) seeded in the semi-IPNs had superior viability and chondrogenesis and osteogenesis capabilities compared to the viability and capabilities of fibrin. We have demonstrated that the preparation of the semi-IPNs under controlled mixing ensured the formation of cell-friendly hydrogels rich in soluble factors and with tunable properties according to the HA MM, rendering them suitable for clinical applications in regenerative medicine.

1. Introduction Leukocyte- and platelet-rich plasma (L-PRP) consists of a concentrate of platelets, leukocytes, proteins and other components that after activation forms fibrin network, a matrix that acts as a reservoir of soluble factors that orchestrate healing mechanisms [1]. Platelets store many growth factors (GFs), chemokines and proteins in their alpha granules, as well as exosomes and microparticles that act as mediators in various physiological processes. Activated leukocytes secrete cytokines that, in addition to their inflammatory behavior, have important roles in maintaining homeostasis by stimulating cell activity [2–4]. Furthermore, the inflammatory phase is crucial for the healing process because it promotes remodeling and induces the tissue contraction phase. Finally, monocyte differentiation into macrophages during these immune response processes is of great importance due to macrophage plasticity into the M1 (inflammatory) and M2 (anti-inflammatory)

⁎

phenotypes and its contribution to regeneration [5,6]. Therefore, both platelets and leukocytes are essential for global regenerative processes [7]. The effectiveness of locally injected L-PRP on tissue regeneration has been observed in clinical studies, particularly in the treatment of cartilage and bone diseases [8–10]. L-PRP efficacy can be potentially increased when it is combined with hyaluronic acid (HA) [11], a glycosaminoglycan present in the extracellular matrix (ECM) of joint tissues in the high molar mass (HMM) form (> 1000 kDa), where it contributes to the maintenance of tissue integrity by promoting its organization, elasticity, lubrication and shock absorption capacity [12,13]. In the organism, HMM HA is naturally degraded into smaller fragments that form oligosaccharides (< 20 disaccharides) and the HA of low (LMM) and intermediate molar mass (20 to 450 kDa) that have different biological properties, such as promoters of angiogenesis and as stimulators of inflammatory cytokine expression [10–12].

Corresponding author. E-mail address: [email protected] (M.H.A. Santana).

https://doi.org/10.1016/j.msec.2019.110547 Received 7 June 2019; Received in revised form 6 December 2019; Accepted 11 December 2019 Available online 13 December 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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homogenized for 30 min at 8 rpm at 25 °C in a Phoenix Luferco homogenizer AP22 (Phoenix Luferco, Araraquara, SP, Brazil). Prior to the preparation of the semi-IPNs, autologous serum containing thrombin was prepared by collecting 5 mL of whole blood from the same donors from whom the plasma was obtained in a VACUETTE tube with a serum clot activator (Greiner Bio-One, Kremsmünster, Austria) and centrifuged for 15 min at 2000 ×g at 25 °C. The serum was separated and mixed with 10% (w/v) calcium chloride at a volumetric ratio of 9:1 of serum to CaCl2 (Sigma-Aldrich, St Louis, USA). Fibrinogen activation and semi-IPN formation were achieved by adding serum and Ca2+ to the homogeneous L-PRP-HA mixture at a concentration of 20% (v/v). After fibrin and semi-IPNs formation, the concentration levels of platelets and leukocytes in the remaining plasma were quantified using the hematologic analyzer. The difference between the concentrations of the blood components in the L-PRP sample and in the remaining plasma was calculated to determine the number of entrapped platelets and leukocytes in the hydrogels. The experiment was performed in triplicate (n = 3) for each condition.

In general, the clinical protocols are based on the sequential injection of L-PRP and HA on the assumed basis that the movement of the patient's joint ensures effective mixing of these compounds [11,14,15]. Due to the high viscosity of concentrated HA solutions, poor mixing between L-PRP and HA could result in nonhomogeneous hydrogels with weak interaction capacity that might not favor cell attachment and proliferation [16]. In addition, the formation of different structures may result in different biological responses, making the comparisons of results difficult. A semi-interpenetrating polymer network (semi-IPN) is defined as a network composed of polymers in both cross-linked and linear forms [17–19]. A fibrin-HA semi-IPN structure is formed by polymerized fibrin and HA coils entangled among the fibers, binding specific sites in fibrin through physical cross-linking but without covalent bonds, and with variable packing arrangements and viscoelasticity, depending on the HA MM and concentration [18,20,21]. Previous studies have shown the capacity of fibrin from P-PRP (poor-leukocyte PRP) mixed with noncrosslinked HA to form hydrogels capable of supporting cell proliferation and differentiation [21,22]. However, a leukocyte-rich formulation might result in a network with novel packing and other properties that have not yet been studied. Therefore, in this work, we prepared fibrin-HA semi-IPNs by mixing L-PRP with LMM HA (~15 kDa) or HMM (> 2000 kDa) under controlled conditions. For the L-PRP preparation, we used a protocol previously established by our group to obtain known ranges of platelet and leukocyte concentration [23]. The fibrin-LMM HA and fibrin-HMM HA semi-IPNs were characterized according to their polymerization kinetics, morphology, swelling patterns, stability, rheological behavior, and the levels of soluble factors released. The capability of the semiIPNs to support cell viability and differentiation was evaluated by using mesenchymal stem cells derived from human adipose tissue (hAdMSCs) seeded in the semi-IPNs and stimulated to chondrogenesis and osteogenesis. The results obtained here are valuable for standardizing protocols to produce semi-IPNs from autologous L-PRP and non-crosslinked HA, with tunable properties according to the HA MM, which can be useful in clinically for regenerative medicine.

2.3. Polymerization time and fiber characterization Fibrin polymerization kinetics was determined by measuring the optical densities of the fibrin and fibrin-HA gels at a 450-nm wavelength for 60 min, with time zero corresponding to the time that the serum and Ca2+ were added, as measured in a FilterMax F5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). From the obtained kinetic profiles, the point of polymerization initiation was established as the point at which the optical density curve first changed slope. The radii and mass/length ratios of the fibrin fibers were characterized using a modified Carr's equation (Eq. (1)) [24].

2π 3Ciμ tλ5

=

( ) (λ dn2 dC

44 15

2

−

184 2 2 2 πr i 154

)

N

(1)

where t is solution turbidity; C is the initial fibrinogen concentration (g/ cm3); N is Avogadro's number; λ is the incident wavelength (cm) (340, 450, 500, 540, 600 and 640 nm); μ = mf/L, with mf as the protein mass (Da) in a fiber of length L (cm) and radius r (cm); and i is the refractive index (1.33). Next, tλ5 was plotted against λ2, resulting in a straight line with a slope that indicated the mass/length ratio, and the ordinate at the origin referred to the square of the average radius. Turbidity was calculated based on its relationship with the optical density (OD) of the fibrin gel, which was measured as the absorbance (Eq. (2)).

2. Material and methods 2.1. Blood collection and L-PRP preparation The use of human specimens in this study was approved by the Ethics Committee of the School of Medical Sciences of Unicamp (Campinas; CAAE: 0972.0.146.000-11). The L-PRP was prepared as previously described [23]. Briefly, whole blood from three different healthy donors was collected after venous puncture in an 8.5-mL tube containing 1.5 mL of anticoagulant acid citrate dextrose solution A (ACD-A) (Vacutainer, BD Biosciences, Allschwil, Switzerland). After collection, 3.5 mL of the anticoagulated blood was transferred to empty 5 mL tubes and centrifuged at 100 ×g for 10 min at 25 °C in a ROTINA 380R centrifuge (Hettich Zentrifugen, Tuttlingen, Germany) with the tubes positioned 45° relative to the rotor. L-PRP, composed of the top and middle layers of the centrifuged blood, was collected and transferred to an empty tube for homogenization and quantification of the components using an ABX Micros ES 60 hematologic analyzer (Horiba ABX Diagnostics, Montpellier, France). Three measurements were made by this equipment.

t = 1 − exp(−OD ·ln(10))

(2)

The experiment was performed in triplicate (n = 3) for each group.

2.4. Scanning electron microscopy (SEM) analysis The morphological and structural characterization of the hydrogels was performed by fixing the fibrin and semi-IPNs in a 4% paraformaldehyde and 2.5% glutaraldehyde solution prepared in PBS and incubating them for 2 h. Then, the samples were dehydrated in graded ethanol dilutions (50, 70, 95 and 100%) at 15 min intervals and dried at the critical point. After gold-coating the gels in a POLARON Sputter Coater, SC7620 (VG Microtech, Uckfield, England), images of the samples were taken using a scanning electron microscope from LEO Electron Microscopy/Oxford (Cambridge, England). The average diameter of the fibrin fibers was determined by measuring 100 individual fibers in three different images using ImageJ software. The elemental composition of the mineralized matrix formed in the hydrogels was characterized by mapping sample surfaces observed using scanning electron microscope-energy-dispersive X-ray spectrometry (SEM-EDS) (LEO Electron Microscopy/Oxford, Cambridge, England).

2.2. L-PRP-HA mixture and semi-IPN preparation The L-PRP was mixed with LMM HA (~15 kDa, according to the manufacturer's instructions, Lifecore Biomedical, Chaska, MN, USA) and HMM HA (> 2000 kDa, according to the manufacturer's instructions, Euflexxa® Ferring Pharmaceuticals, Saint-Prex, Switzerland) at a 1:1 volume ratio to obtain a final HA concentration of 1 mg/mL. To generate a homogeneous mixture, the L-PRP-HA combinations were 2

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2.5. Determination of fibrinogen concentration

in the linear viscoelastic region (LVE) with the strain amplitude (γ0 = 1%) in the angular frequency range of 0.1 to 100 rad/s. The experiments were performed in triplicate (n = 3) for each group. From the viscoelastic (energy storage) and viscous (energy loss) moduli, the complex modulus (G*), and the damping factor (tan(δ)) were calculated by Eqs. (7) and (8).

The concentration of fibrinogen in the L-PRP and in the fibrin network was determined as described in a previously published protocol [25]. After polymerization, the fibrin was centrifuged at 500 ×g for 5 min, and the supernatant was discarded. This step was repeated three times. Then, NaOH (Sigma-Aldrich, St Louis, USA) was added to the tubes containing fibrin, which were left in a water bath at 95 °C for 10 min. Then, the sample was treated with water, NA2CO3 and FolinCiocalteu phenol reagent (Sigma-Aldrich, St Louis, USA) for 30 min, and the absorbance measured at 650 nm. The results were expressed as the fibrinogen concentration, which was calculated using the calibration curve previously prepared with data on commercially available fibrinogen from human plasma (Sigma-Aldrich, St Louis, USA) (R2 = 0.993). The experiment was performed in triplicate (n = 3) for each group.

tan(δ )

G∗ = [(G′)2 + (G″)2]1/2

Vd

Swelling ratio =

Mass loss (%) =

× 100

(3)

Ws − Wd Wd Wf − Wd Wd

2.10. Quantification of GFs and cytokines release kinetics Fibrin and semi-IPNs were incubated with and without 1 × 104 hAdMSCs, and the kinetics of the GFs and cytokines release was quantified by collecting all the medium (DMEM) at 3, 8, 24 and 72 h and storing it at −80 °C, with new medium added to maintain well volume. The cumulative concentration levels of GFs (TGF-β1, PDGF-AB and VEGF) and cytokines (IL-1 β, IL-6, and TNF-α) were quantified using a Bioplex Pro kit with a Bioplex 200 system (Bio-Rad, Hercules, USA). Experiments were performed in triplicate (n = 3) for each condition.

(4)

× 100

(5)

2.7. FTIR analysis The degraded hydrogels were lyophilized in an L-101 lyophilizer (Liotop, São Carlos, Brazil) for two days. The FTIR analyses on the dried samples were performed with a Thermo Scientific Nicolet 6700 spectrophotometer (Thermo Fisher Scientific, Madison, USA), using the single-reflection germanium attenuated total reflection (ATR) technique, in a Smart OMNI-Sampler (Thermo Fisher Scientific, Madison, USA). The wavelength range of the instrument was 4000–750 cm−1 with a resolution of 4 cm−1.

2.11. Assessment of h-AdMSC viability Cell viability was determined by live/dead assay over two weeks using a LIVE/DEAD cell imaging kit (Thermo Fisher Scientific, Waltham, MA, USA). At predetermined days, the medium was removed from the wells, and the IPNs were washed with PBS. Then, 200 μL of the LIVE/DEAD reagent was added to the wells, and the samples were incubated for 30 min. After this period, images of the samples were taken with a confocal microscope (Leica Microscope TCS SP5 II, Wetzlar, Germany). The number of living and dead cells was calculated by counting cells from at least three different images using ImageJ software.

2.8. Rheological characterization The rheology of the L-PRP, L-PRP-HA mixtures, fibrin and the semiIPNs was studied in an Anton Paar MCR-102 Modular Compact Rheometer (Anton Paar, Graz, Austria). The tests were conducted using a cone-plate geometry (CP50-1) of 50 mm in diameter, a cone angle of 0.9815° and a truncation of 0.97 μm. Steady-state shear tests were performed in the range of 0.01 to 1000 s−1 at 25 °C. Experimental data were fitted using the power-law model, which is defined by Eq. (6).

τ = mγ̇n

(8)

h-AdMSCs from human subcutaneous adipose tissue were acquired from patients who had undergone abdominal liposuction surgery or lipo-aspiration at the University Hospital. Cells were isolated and cultured as previously described [26]. The precultured cells were trypsinized and resuspended in the L-PRP and L-PRP-HA mixtures to obtain 1 × 104 cells/hydrogel. After homogenization, 160 μL of L-PRP and LPRP-HA with h-AdMSCs were added to a 48-well plate and activated with 40 μL of serum and Ca2+. Once the gels were formed, 750 μL of low-glucose DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 1% of penicillin/streptomycin was added to the wells, and the cells were cultured in a 37 °C incubator with 5% CO2. The medium was changed every three days. The culture medium was not supplemented with FBS due to the high capacity of L-PRP to provide nutrients for cell survival and growth [27]. To induce chondrogenesis and osteogenesis, 3 × 105 cells per hydrogel were cultured for 3 to 4 weeks in 750 μL of solution from a StemPro™ osteogenesis and chondrogenesis differentiation kit (Thermo Fisher Scientific, Waltham, USA). The cells cultured in regular DMEM constituted the control for the differentiation assay. The medium was changed every three days.

The porosity of the networks was determined by soaking the dried samples in ethanol and measuring the ratio of the void volume (Vp) to the volume of the dried hydrogel (Vd), as shown in Eq. (3). Pore sizes were determined by measuring at least 100 pores in 3 different images using ImageJ software. To assess the swelling behavior of the hydrogels, dried fibrin and semi-IPNs were weighed (Wd) and dipped in phosphate-buffered saline (PBS, pH 7.4 at 37 °C) until they reached equilibrium. At predetermined times, the hydrogels were weighed (Ws), and the swelling ratio was calculated using Eq. (4). The swollen hydrogels were weighed (Wf), and the mass loss was calculated according to Eq. (5). The experiments were conducted in triplicate (n = 3) for each group.

Vp

(7)

2.9. h-AdMSCs isolation and culture in fibrin and semi-IPNs

2.6. Porosity, swelling behavior and hydrolytic degradation characterization

Porosity (%) =

G″ G′

2.12. Gene expression analysis Quantitative real-time PCR (RT-PCR) was used to analyze the gene expression profiles of the h-AdMSCs cultured in fibrin or semi-IPNs for 3 weeks. Initially, RNA from each sample was extracted by using an RNAspin mini kit (GE Healthcare Life Sciences, Chicago, USA), and cDNA was transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems, Abingdon, RU) in a PTC-100 PCR thermo

(6)

where τ is the shear stress; m the consistency index; γ̇ is the shear rate; and n is the power-law index. The oscillatory measurements were taken 3

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cycler (MJ Research Inc., Waltham, MA). Subsequently, RT-PCR was performed in a 7500 real time PCR system (Applied Biosystems, Foster City, USA) using SYBR Green dye. The reaction volume comprising the SYBR Green dye, forward and reverse primers and the cDNA template was set to 12 μL. The analysis condition was set as 55 °C for 2 min followed by 95 °C for 1 min, 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The expression levels of chondrogenesis-specific genes, Sox9, aggrecan, collagen I and collagen II, and osteogenesis-specific genes, collagen I, collagen X, and osteocalcin, were quantified. The results were analyzed using the comparative 2−ΔΔCT method, with the expression of genes normalized to that of the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase (HPRT), and to the respective controls (samples cultured in regular medium). The experiment was performed in triplicate (n = 3) for each group using the L-PRP obtained from the whole blood of three different donors. The primer sequences are listed in Table S1.

Table 1 Concentration levels of blood components in the whole blood and in L-PRP.

3

3

Platelets × 10 /mm Total leukocytes × 103/mm3 Lymphocytes × 103/mm3 Monocytes × 103/mm3 Granulocytes × 103/mm3 Erythrocytes × 106/mm3

Whole blood

L-PRP

181 ± 5 4.30 ± 0.10 1.47 ± 0.06 0.10 ± 0.00 2.73 ± 0.06 3.37 ± 0.04

294 ± 1 1.20 ± 0.01 0.87 ± 0.06 0.00 ± 0.00 0.33 ± 0.06 0.20 ± 0.00

3. Results 3.1. Blood and L-PRP composition The concentration levels of blood components in both whole blood and L-PRP are shown in Table 1. Platelet and leukocyte concentration levels in the L-PRP were 294 ± 1 × 103/mm3 and 1.20 ± 0.01 × 103/mm3, respectively, with a platelet/leukocyte ratio of 245 ± 0.8. The centrifugation conditions enabled the recovery of a relatively high concentration of lymphocytes compared to that of the granulocytes; the lymphocyte/granulocyte ratio was 2.60 ± 0.58.

2.13. Immunocytochemistry After 4 weeks of culture under differentiation stimulus, fibrin and semi-IPNs with the h-AdMSCs were gently washed with PBS (pH 7.4) and fixed in a 4% paraformaldehyde solution for 2 h at 4 °C. The cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St Louis, USA) for 30 min, and nonspecific binding was inhibited using a blocking solution of 5% bovine serum albumin (BSA) (Sigma-Aldrich, St Louis, USA) for 1 h at room temperature. Subsequently, the samples were incubated at 4 °C overnight with the primary polyclonal antibodies to collagen I (1:2000) and collagen II (1:200) (Invitrogen, Carlsbad, USA), followed by a 2 hour incubation at room temperature with the IgG secondary antibody (Alexa Fluor 488, 1:200) (Invitrogen, Carlsbad, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St Louis, USA). The experiment was performed in triplicate (n = 3) for each group using the L-PRP obtained from the whole blood of three different donors. Fluorescence images were taken by a confocal microscope by Z-stacking from 10 to 15 images (Leica Microscope TCS SP5 II, Wetzlar, Germany). Total collagen area was calculated by measuring at least three different images from each donor using ImageJ software.

3.2. Effects of LMM HA and HMM HA on fibrin polymerization and hydrogel formation Initially, the addition of HA to the L-PRP resulted in phase separation, which was more pronounced for the L-PRP-HMM HA (Fig. S1, Supplementary Material). Therefore, the hydrogel mixtures were prepared under controlled conditions (8 rpm, 30 min and 25 °C) to assure the mixture was homogeneous and that uniform hydrogel structures were formed (Fig. 1A). From the microstructural perspective, the effects of both molar masses of HA were initially observed based on the timedependence of the polymerization reaction (Fig. 1B). The polymerization of fibrin was initially very rapid, with the gelation point reached at 3.7 ± 0.9 min. In the presence of both HAs, an increase in the optical density values was observed as was an increase in the polymerization times, which were 6.3 ± 0.8 min for fibrin-LMM HA and 10.5 ± 1.2 min for fibrin-HMM HA (Fig. 1C). The concentration of the fibrinogen in L-PRP was determined as 4.7 ± 0.4 mg/mL. After hydrogel formation, the fibrin was depolymerized with NaOH, and the fibrinogen concentration was measured as 4.6 ± 0.5, 3.2 ± 0.1 and 3.5 ± 0.2 mg/mL for fibrin, fibrin-LMM HA and fibrin-HMM HA, respectively (Fig. 1D). Both fibrin-LMM HA and fibrin-HMM HA showed a high capacity (p < 0.05) to entrap leukocytes (~94%) compared to the capacity of fibrin to entrap leukocytes (87.5 ± 0.5%) (Fig. 1E).

2.14. Histological preparation Fibrin and semi-IPNs containing the h-AdMSCs cultured for 4 weeks were gently washed with PBS (pH 7.4), and fixed in a 4% paraformaldehyde solution for 2 h at 4 °C. Then, the samples were rinsed twice with PBS, dehydrated in 70% ethanol for 7 sequential 30 min cycles and diaphonized in xylol in 3 sequential 30 min cycles. The hydrogels in paraffin blocks were sliced (sections of 5 μm) and maintained in an oven at 60 °C for 24 h. The slices were deparaffinized in xylol and rehydrated in graded ethanol from 100 to 75% and water. The samples cultured under chondrogenic induction conditions were stained with Picro Sirius red for the detection of collagen deposition, and the samples cultured under osteogenic induction conditions were stained with Alizarin red to detect calcium deposition. The samples were scanned by a digital slides scanner (3DHistech, MIDI). The experiments were performed in triplicate (n = 3) for each group using the L-PRP obtained from the whole blood of three different donors. Pixel intensity was measured from at least three different images of each donor sample using ImageJ software.

3.3. Fibrin fiber characterization The SEM images show the structures and morphologies of the fibrin networks, which presented with interconnected nanofiber organization (Fig. 2A). Fibers from the fibrin hydrogel were thin and elongated with an average diameter of 132 ± 35 nm (Fig. 2A(i)), while we observed thicker fibers in both semi-IPN hydrogels that were arranged in an inhomogeneous matrix and in more closely packed domains, and with the average fiber diameter of 190 ± 40 and 225 ± 50 nm for fibrin-LMM HA and fibrin-HMM HA, respectively (Fig. 2A(ii) and (iii)). The diameter estimative obtained by using the modified Carr's equation were consistent with those measured in the SEM images; they were at the same order of magnitude and showed that fibers in the fibrin-HA hydrogels were thicker than those in the fibrin. However, a smaller difference was found between the diameter of the fibers from fibrin and the fibrin-HA hydrogels compared to the values obtained from the SEM images (Fig. 2B). The results from the Carr's equation showed that fibers in the fibrin-HA hydrogels were shorter, indicating that HA at both MM

2.15. Statistical analysis The results are presented as the mean ± standard deviation (SD). When relevant, one-way analysis of variance (ANOVA) with a Tukey's test was used for statistical analysis. A 95% confidence level was considered significant (p < 0.05). 4

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Fig. 1. Fibrin and fibrin-HA formation. (A) Images from before and after polymerization showing (i) L-PRP and fibrin, (ii) L-PRP-LMM HA and fibrin-LMM HA and (iii) L-PRP-HMM HA and fibrin-HMM HA. Scale bar = 0.5 cm. (B) Polymerization kinetics monitored by optical density. (C) Gelation points were determined based on changes in the slope of kinetics curves. (D) Concentration of polymerized fibrinogen to form fibrin fibers. (E) Percentage of total platelets and leukocytes entrapped within the networks. *p < 0.05.

levels prevented fiber elongation, thus favoring lateral polymerization (Fig. 2D). The porosity of the networks was approximately 60% and was significantly higher for fibrin (63.6 ± 1.4%) than it was for fibrin-LMM HA or fibrin-HMM HA (59.3 ± 1.0 and 56.0 ± 1.1%, respectively) (Fig. 2C). Consequently, larger pores were observed in the fibrin, with the size decreasing with increases in the HA MM (Fig. 2D).

hydrogels (Fig. 3B). The formation of semi-IPNs was investigated by FTIR analysis of the degraded hydrogels (Fig. 3C). The presence of the characteristic HA peaks was observed, with the large peak at 3300 cm−1 related to aqueous OeH stretching bands overlapping the peak of the NeH groups at 3100 cm−1. The peaks with low intensity at 2900 cm−1 correspond to CeH symmetrical and CeH2 asymmetrical stretching. The C]O amide stretching band at 1650 cm−1 overlaps with the peak corresponding to carboxyl CeO asymmetrical stretching at 1615 cm−1. The stretching band of the symmetrical carboxyl CeO is shown at 1410 cm−1, and the peak at 1045 cm−1 is attributed to C-OH stretching. To highlight the interactions between fibrin and HA in the semi-IPNs, a second derivate of the absorbance intensity was calculated, and it showed an increase in intensity at 1550 cm−1 and 1650 cm−1 in the semi-IPN spectra, which corresponded to the NeH stretching of the amide II and amide I in fibrin, respectively (Fig. S2, Supplementary Material).

3.4. Physical and structural characterization of the hydrogels Rapid swelling was observed for the three hydrogels, which showed the tendency to stabilize in the second hour for fibrin, whereas both the fibrin-HA hydrogels continued to swell for up to 4 h, when they reached equilibrium. A significantly higher swelling ratio (p < 0.05) was observed for fibrin-HMM HA compared to both fibrin-LMM HA and fibrin after 4 h, while fibrin swelling was significantly lower than semi-IPN swelling after 2 h (Fig. 3A). Hydrolytic degradation showed continuous mass loss with time, which tended to stabilize by day 10. No significant difference was observed between the fibrin and fibrin-HA hydrogels, with the mass loss being < 20% in the first 2 weeks, confirming the stability of the

3.5. Rheological behavior of fibrin and semi-IPNs The rheological properties of the pre-polymerized hydrogels were 5

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Fig. 2. The morphology characterizations of the fibrin fibers and their networks. (A) SEM images of (i) fibrin, (ii) fibrin-LMM HA and (iii) fibrin-HMM HA, at 3 kX and 10 kX magnitude, with the fiber diameter distribution histograms highlighted. White, yellow and blue arrows point to platelets, erythrocytes and leukocytes, respectively. (B) Characterization of the fibers by Carr's equation as the diameter and mass/length ratio. (C) Network porosity. (D) Pore size as measured using ImageJ software. *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

associated with the viscoelastic and viscous characteristics, respectively. For L-PRP and L-PRP-LMM HA, a weak solid-like (G′ > G″) behavior was observed, while for L-PRP-HMM HA, a tendency towards transition to a liquid-like behavior (G″ > G′) was verified at high frequencies (Fig. 4C). After hydrogel crosslinking, the curves confirmed their viscoelastic behavior, with G′ decreasing as MM HA increased, dropping from ~100 Pa for fibrin, to ~40 Pa and ~10 Pa for fibrin-LMM HA and fibrin-HMM HA, respectively (Fig. 4D). In addition, the complex modulus G*, which depicts the stiffness properties of the biomaterials showed the low contribution of the viscous moduli G" (G* ~ G'), as indicated by the following descending values. The results from the damping factor tan(δ) analysis showed that the hydrogels exhibited

evaluated by steady-state shear characterization, in terms of shear stress (Fig. 4A) and viscosity (Fig. 4B) as a function of the shear rate. The curves revealed Bingham type fluid behavior with slight differences between L-PRP-LMM HA and L-PRP. On the other hand, L-PRP-HMM HA had increased yield stress and viscosity values, and also it presented an increased shear-thinning behavior, as revealed when the experimental data were fitted using the Ostwald–de Waele power-law model. An increase in the consistency index (m) value was observed for L-PRPHMM HA compared to that for L-PRP-LMM HA and L-PRP, while the power-law index (n) values were < 1 in all cases (Table S2, Supplementary Material). The viscoelastic behaviors of samples before and after fibrin polymerization were characterized through frequency sweep tests, in which the storage (G′) and the loss (G″) moduli were 6

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Fig. 3. Physical characterization of fibrin and the fibrin-HA hydrogels. (A) Swelling pattern and (B) hydrolytic degradation. (C) FTIR spectra for LMM-HA, HMM-HA, and the semi-IPNs of fibrin-LMM HA and fibrin-HMM HA dried after 2 weeks of incubation, showing the characteristic bands of HA (highlighted in gray) and bands resulting from fibrin-HA interactions (circled in orange). The spectra were vertically shifted to avoid overlapping. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.7. Effects of the semi-IPNs on chondrogenesis and osteogenesis of the hAdMSCs

lower values than the respective pre-polymerized hydrogels, for which the values were close to 1 (Fig. S3, Supplementary Material). From our results, we can assume that new matrix structures were formed through the connections made among the different components, which were strongly supported by the fibrin-HA interactions. Semi-IPNs possessed different viscoelasticity and network packing, which was in the range of micrometers (Fig. 5).

RT-PCR was performed after 21 days of culture and showed that, for cells induced to chondrogenesis, the expression of Sox9 was significantly higher in the fibrin-HMM HA (Fig. 7A), while the level of aggrecan expression was significantly higher (p < 0.05) in both the semi-IPNs compared to that in fibrin (Fig. 7B). The collagen II/collagen I ratio was calculated and was higher in fibrin-HMM HA (0.78 ± 0.07) than it was in fibrin (0.34 ± 0.04) and fibrin-LMM HA (0.0002 ± 0) (Fig. 7C). The expression levels of the osteogenic genes collagen types I and X were significantly higher (p < 0.05) for the h-AdMSCs in the semi-IPNs (Fig. 7D and E), particularly compared to the levels in fibrinHMM HA, while no significant difference among the samples was observed for osteocalcin expression (Fig. 7F). Deposition of ECM was evaluated by immunocytochemistry and histological analysis after 28 days of culture, and the evidence indicated higher secretion levels of the specific chondrogenic marker collagen II (Fig. 7G, H and S4A, Supplementary Material) from the cells in fibrinHMM HA, corroborating the gene expression analysis. Similarly, Picro Sirius red staining indicated greater collagen deposition by cells in the semi-IPNs composed of HMM HA (Fig. S5A, S5B and S5E, Supplementary Material). The deposition levels of the osteogenic marker collagen I were not significantly different between fibrin and the semi-IPNs (Figs. 7I, J and S4B, Supplementary Material), while the deposition level of the calcified matrix as indicated by Alizarin red staining was found to be significantly higher (p < 0.05) in the semi-IPNs compared to that in fibrin (Fig. S5C, S5D and S5F, Supplementary Material). SEMEDS analysis confirmed the presence of calcium and phosphorous, the main components of hydroxyapatite, in the mineralized matrix formed in the hydrogels cultured under differentiation stimulus (Fig. S6, Supplementary Material).

3.6. h-AdMSCs response to fibrin and semi-IPNs At the molecular level, the results showed an increased concentration levels of anabolic GFs released from fibrin compared to the respective concentration levels released from the semi-IPNs, while a more pronounced release of inflammatory cytokines was observed for both fibrin-HA hydrogels (Fig. 6). Interestingly, the levels of PDGF-BB (Fig. 6A) and TNF-α (Fig. 6D) concentration were higher for the hydrogels without h-AdMSCs. After 24 h, the concentration levels of the TGF-β1 (Fig. 6B) and VEGF (Fig. 6C) released from the hydrogels containing cells increased substantially, with the concentration being more pronounced for fibrin. In contrast to the findings for TGF-β1, the release of the angiogenic factor VEGF from fibrin-LMM HA was observed to be increased compared to the concentration released from the fibrin-HMM HA. High levels of inflammatory cytokines IL-1β (Fig. 6E) and IL-6 (Fig. 6F) were secreted from the hydrogels with the h-AdMSCs after the first hours of incubation and stabilizing within 24 h. The confocal microscopy images showed that on the 7th day of culture, the cells presented with a spreading phenotype in the semiIPNs compared to those in the fibrin, in which most cells had a round shape (Fig. 6G and H). In addition, the h-AdMSC viability was significantly higher in the semi-IPNs from day 7 through two weeks in culture (Fig. 6I).

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Fig. 4. Steady-state shear characterization of the L-PRP and L-PRP-HA pre-hydrogels by (A) shear stress and (B) viscosity, showing that HMM HA significantly affected the rheological behavior of the L-PRP. Variations in G′ and G″ with angular frequency of (C) pre-hydrogels: (i) L-PRP, (ii) L-PRP LMM HA and (iii) L-PRP HMM HA and (D) hydrogels: (i) fibrin, (ii) fibrin-LMM HA and (iii) fibrin-HMM HA.

Fig. 5. Schematic representation of the pre-hydrogels and hydrogels microenvironments, showing the structural differences of fibrin networks and the semi-IPNs. 8

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Fig. 6. Kinetics of the GFs and cytokines release from fibrin and the semi-IPNs with and without h-AdMSCs. (A) PDGF-BB, (B) TGF-β1, (C) VEGF, (D) TNF-α, (E) IL-1 and (F) IL-6. (G) Schematic illustration of h-AdMSCs in the three microenvironments. (H) LIVE/DEAD images taken on day 7 of the cells with (i) fibrin, (ii) fibrinLMM HA and (iii) fibrin-HMM HA. (I) Viability of the h-AdMSCs cultured in fibrin and the semi-IPNs for 2 weeks.

4. Discussion

an optimal PRP formulation, due to the low concentration or the total absence of leukocytes [29,30], the L-PRP composition greatly contributes to the inflammatory phase and the M1 into M2 macrophage polarization, which play important roles in homeostatic regulation and tissue regeneration mechanisms [6,7,31].

L-PRP is a concentrate of platelets and leukocytes that has been clinically applied in the treatment of injured cartilage and bone, where it showed positive responses [9,10,28]. Although P-PRP is considered 9

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Fig. 7. Assessment of the chondrogenic and osteogenic differentiation potential of h-AdMSCs cultured in fibrin and the semi-IPNs. Results of the RT-PCR analysis of the chondrogenic gene levels of (A) Sox9 and (B) aggrecan and (C) the collagen II/collagen I ratio. Results from the RT-PCR analysis of the osteogenic genes (D) collagen I, (E) collagen X and (F) osteocalcin. (G) Results from the immunocytochemical analysis for the chondrogenic marker collagen II in (i) fibrin, (ii) fibrin-LMM HA and (iii) fibrin-HMM HA. (H) Total collagen II area calculated using ImageJ software. (H) Results from the immunocytochemistry analysis for the osteogenic marker collagen I in (i) fibrin, (ii) fibrin-LMM HA and (iii) fibrin-HMM HA. (I) Total collagen I area calculated using ImageJ software. *p < 0.05.

while inside the syringe [32]. However, the mixture should be injected in the patient a few seconds before the gelation point is reached to prevent leakage at the site of the injury. The presence of HA was also able to entrap relatively more leukocytes within the fibrin-HA structures, which could affect biological responses [33]. Slight declines and oscillation curves were observed during fibrinHA gelation, indicating a possible reorganization of HA among the fibers [34]. The increase in turbidity of the semi-IPNs was directly associated with the increase in the fibrin fiber diameter and mass/length ratio [24]. Due to specific binding between HA and fibrinogen, vertical alignment and fiber elongation were inhibited, resulting in shorter and

Here, we mixed L-PRP with non-cross-linked LMM HA and HMM HA to form semi-IPN hydrogels through the physical cross-linking of fibrin and globular HA. We observed a significant delay in fibrin polymerization in the presence of both HA types, either by preventing some fibrinogen monomers from reacting or by inducing lateral polymerization, which is slower than longitudinal. Due to the increased chain size of the HMM HA, larger coils were formed, which contributed to the slower protofibrillation process compared to that for LMM HA. The slower gelation point of fibrin-HA is of interest for clinical use; it would enable physicians enough time to administer the L-PRP-HA mixture and prevent the preparation from reaching the gelation point 10

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the fibrin network, and differences in topology greatly influence cell adhesion and macrophage polarization, thus contributing to faster and more effective regeneration [46]. The kinetics of GFs and cytokines release showed that the concentration levels of the anabolic factor PDGF-BB and the catabolic factor TNF-α were higher in the absence of h-AdMSCs, indicating that they were exclusively secreted by activated platelets and leukocytes. We observed that h-AdMSCs did not produce, but responded to PDGFBB and TNF-α, being activated and stimulated to release GFs TGF-β1 and VEGF after 24 h of incubation. Despite being a potent inflammatory factor, TNF-α also activates MSCs through different signaling pathways, playing important roles in modulating cell functions [2–4]. It was interesting to note that the concentration of the GFs released from the semi-IPNs was significantly lower than that from fibrin as a result of the networks packing, which acted as a diffusive barrier. Higher levels of VEGF were secreted from fibrin-LMM HA than were secreted from fibrin-HMM HA at 72 h, suggesting the angiogenic potential of this semiIPN. Both LMM HA and HMM HA had crucial roles in leukocyte activation by binding to TLR4/TLR2 and CD44 receptors, respectively [47,48], and by stimulating the secretion of the inflammatory cytokines IL-1β and IL-6. This response was less pronounced for the semi-IPNs composed of HMM HA, which is known for its anti-inflammatory character, due to the CD44 clustering that protects cells from the binding of inflammatory mediators [49]. However, this HA-CD44 binding also activates leukocytes and stimulates cytokine release [48], as observed here. In the presence of h-AdMSCs, the concentration of IL1 β and IL-6 increased, indicating that the h-AdMSCs were also activated and stimulated to secrete cytokines, suggesting that an inflammatory phenotype was acquired by the cells [50]. Despite the inflammatory microenvironment, the semi-IPNs were favorable for h-AdMSC viability, indicating that there was an important anabolic/catabolic balance provided by the PDGF-BB and TNF-α released from the platelets and leukocytes, and along with the hydrogel properties, they modulated cell responses [51]. This result was observed in the results of the by LIVE/DEAD assay, which showed that the h-AdMSCs had an elongated phenotype when cultured in the semi-IPNs, while in fibrin, the cells presented a more rounded shape. This difference was likely due to the new packing structure of the semi-IPNs, which formed relatively less porous networks with different stiffness, which affected the cell shape [40]. In addition, this outcome could also be indicative of cell preference for differentiation in these hydrogels. To confirm the h-AdMSC differentiation potential, chondrogenesis was assessed after 21 days in culture by using the specific markers Sox9, which is crucial in the early stages of differentiation, and aggrecan and collagen II, which are present in mature cartilage. The results showed that fibrin-HMM HA favored the expression of Sox9, and both semiIPNs had high levels of aggrecan expression compared to that of fibrin. To commit to the chondrogenic lineage, the collagen II/ collagen I ratio is expected to be equal or > 1 [52], which was close to the value obtained for the fibrin-HMM HA. On the other hand, fibrin-LMM HA downregulated collagen II expression. We then performed histological and immunocytochemical assays on the samples cultured for 28 days, and because collagen II deposition was observed, we concluded that fibrin-LMM HA had less capacity to stimulate h-AdMSCs to chondrogenesis in a shorter time compared to the capacity of fibrin-HMM HA to promote chondrogenesis [53]. The fibrin-HMM HA semi-IPNs also showed significantly higher expression of the specific osteogenic markers collagen I and collagen X, genes that are expressed prior to calcification. Osteocalcin, a mineralization-related gene, which is expressed in late osteogenesis, was expressed at high levels in all three samples, as also found in the matrix deposition analysis. Even though the semi-IPNs were softer than fibrin, our results showed high h-AdMSCs differentiation potential in the fibrin-HA microenvironment, especially fibrin-HMM HA. However, further studies should be performed by incorporating a stiffer biomaterial

thicker fibers [34]. In addition, denser networks were formed for the semi-IPNs, being more pronounced for fibrin-HMM HA due to the larger coils and higher number of junctions and chains overlaps, as compared to that of LMM HA. Consequently, denser mesh-like network with lower porosity was observed for fibrin-HMM HA structure, even though the amount of HA was the same for both semi-IPNs. Fibers alignment was random and did not follow a specific direction, which needs an external stimulus [35]. Although the difference between fiber thickness in fibrin and the semi-IPNs was less sensitive according to Carr's equation than the calculations based on the SEM images, the method based on Carr's equation was shown to be suitable for estimating changes in fiber diameter and length. Physical characterization analysis revealed that, because of the increased hydrophilic domains in the semi-IPN structure, a significantly higher swelling ratio was observed for fibrin-HAs compared to that found in fibrin, indicating that semi-IPNs would be favorable for nutrient diffusion. A hydrolytic degradation assay was conducted in PBS at 37 °C to simulate the microenvironment in vivo. The results showed that < 20% of the hydrogel mass was lost in the first two weeks, indicating that the semi-IPNs presented moderate structural stability, which we assume to be favorable for sustaining h-AdMSC in vivo for a long time after injection. The results from the FTIR analysis showed that specific bands of HA were present in the semi-IPN structures after two weeks of induced degradation [36]. Therefore, we concluded that HA did not diffuse throughout the networks over time. The rheological behavior indicates the pre-hydrogels are yield stress materials, which need an applied stress for starting plastic deformation. The yield stress effect was more pronounced for L-PRP HMM HA as compared to L-PRP or L-PRP LMM HA. These results indicate that initially, the molecular interactions of the dispersed components form a network, which is susceptible to breakage during the material's flow. In this case, the viscosity decreases with the shear rate, characterizing the shear thinning behavior, which is typical of an entangled network, and, here, was defined by the presence of the HA chains and their interactions with the other components [37], being more pronounced for the larger HA molecules. These results reflects the specific interactions between fibrinogen and HA during homeostasis, inflammation and wound healing [38]. The increased shear-thinning behavior and the longer gelation time make L-PRP-HMM HA a favorable matrix for clinical applications. In addition to the available time for preparation, it has greater capacity to act as a fluid under high shear stress (during syringe pressure) and as a solid under low shear stress (after injection), which helps prevent leakage at the site of the injury [39]. To confirm this behavior, injectability tests of the pre-hydrogels will be performed in our future in vivo studies. Hydrogel stiffening was affected by the presence of HA at both MM levels, and the semi-IPNs formed less-stiff hydrogels compared to those of fibrin. According to the values of the complex modulus G*, HA decreased fibrin stiffness, especially HMM HA. G* was similar to the elastic modulus G', indicating the energy was mostly stored than lost, due to the packing of the matrices. As reported in most non-autologous scaffolds, substrate stiffness modulates cell attachment, proliferation and differentiation to a degree-dependent on the stiffness of the native tissue [40,41]. Substrate stiffness has been directly implicated in inflammation, once it promotes leukocyte transendothelial migration [42]. The stiffness level reported in the literature for osteogenic differentiation in terms of G* is in the order of 1.5 kPa or greater [43,44]. We observed that the semi-IPNs produced here were softer, with G* in the order of 10 to 100 Pa, which could lead cells to a poor differentiation capacity. However, in addition to stiffness, chemical factors, crosslink agents and surface topology are also crucial to stimulate cell behavior [45]. As the semi-IPNs prepared here carry high numbers of soluble factors, the biochemical factors should promote an intense stimuli to stem cells, in which we hypothesize that the stimuli provided by substrate stiffness and other mechanical stimuli would be secondary. In addition, surface properties, such as an increased area, as provided by 11

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in the semi-IPNs to obtain a hydrogel that is stiff, autologous and rich in soluble factors to investigate the benefits for cartilage and bone engineering.

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5. Conclusions This study demonstrated the feasibility of the preparation of autologous and cell-friendly semi-IPNs composed of fibrin from L-PRP and non-cross-linked HA. The hydrogel properties were tunable with the HA MM, indicating that a wide range of HA MM levels can be used to adjust the semi-IPN properties according to the needed application. Similarly, the concentration of the soluble factors could also be adjusted to obtain an optimal anabolic/catabolic balance. Despite the semi-IPNs forming softer hydrogels compared to fibrin, h-AdMSCs preferably differentiated in these microenvironments, particularly in fibrin-HMM HA, showing the potential of these semi-IPNs to be used in regenerative medicine.

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Credit authorship contribution statement

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Bruna Alice Gomes de Melo: Writing - original draft, Methodology, Investigation, Formal analysis. Carla Giometti França: Investigation. José Luis Dávila: Investigation. Nilza Alzira Batista: Investigation. Carolina Caliari-Oliveira: Writing - review & editing. Marcos Akira d'Ávila:Investigation. Ângela Cristina Malheiros Luzo: Conceptualization. José Fabio Santos Duarte Lana: Conceptualization. Maria Helena Andrade Santana: Methodology, Supervision, Writing - review & editing.

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Declaration of competing interest [17]

The authors declare no conflicts of interest. [18]

Acknowledgment The authors gratefully acknowledge Prof. Dr. William Dias Belangero for the use of the BAL-TEC CPD 030 dryer and for the assistance with the histological preparations, and Dr. Adriana da Silva Santos Duarte for the helpful assistance with the h-AdMSC isolation and culture. This work was supported by The São Paulo Research Foundation (FAPESP), grant numbers 2015/23134-8 and 2016/101320.

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Appendix A. Supplementary data [22]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110547. References

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