Porous Poly(vinyl alcohol)-Based Hydrogel for Knee Meniscus

Article Cite This: ACS Biomater. Sci. Eng. 2018, 4, 1518−1527

Porous Poly(vinyl alcohol)-Based Hydrogel for Knee Meniscus Functional Repair Luca Coluccino,†,‡,§ Riccardo Gottardi,†,§,⊥ Farouk Ayadi,∥ Athanassia Athanassiou,‡ Rocky S. Tuan,§ and Luca Ceseracciu*,# ‡

Smart Materials and #Materials Characterization Facility, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy Department of Orthopaedic Surgery, Department of Chemical Engineering, and the McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, United States ⊥ Ri.MED Foundation, Palermo 90133, Italy ∥ UNIROUEN, INSA Rouen, CNRS, PBS, Normandie Universite, 76000 Rouen, France

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ABSTRACT: The meniscus has a key role within the knee joint, conferring stability, absorbing and redistributing loads, and influencing the overall movement proprioception. Recent developments in the treatment of meniscal injury have progressively shifted the focus from general resection to functional repair, with the recognition that restoring the biomechanical meniscal function helps to prevent degenerative changes in the knee joint and the insurgence of osteoarthritis. To address this clinical need, we have developed a biomimetic implant based on a porous poly(vinyl alcohol) (PVA) hydrogel. Such hydrogels are stable, biocompatible, and suitable to surgical translation, and their mechanical properties can be tuned to reduce the mismatch in the case of partial meniscectomy. The PVA implant structure is porous and permeable, allowing fluid flows and facilitating anatomical integration in situ. Here, we present a chemo-physical characterization of PVA porous hydrogels, focusing on their tunable morphology and associated viscoelastic properties. Biocompatibility was evaluated using primary bovine meniscal fibrochondrocytes, and integration with native tissues was assessed in an ex vivo model. Overall, our results suggest that a synthetic meniscal implant based on a porous PVA hydrogel could restore the physiological function of the meniscus and represent a promising clinical alternative to current resection treatments. KEYWORDS: PVA hydrogel, meniscus repair, fibrocartilage, biomaterial, prosthetic implant

1. INTRODUCTION

other chronic, degenerative joint diseases. The restoration of meniscal functions with a biostable substitute able to mimic the meniscus biomechanical native properties is a possible route to avoid these problems.12−18 Total meniscal replacement allografts are a possible treatment, but there are limitations in terms of clinical availability, size tenability, and costs. Thus, a valid alternative could be represented by a synthetic implant combining long-term durability, clinical processability, and tunable biomechanical properties that resemble those of the natural meniscus. In the work reported here, we have developed

The meniscus fibrocartilage plays a key role in knee joint biomechanics in terms of load transmission, shock absorption, and general proprioception.1−4 The menisci mainly transfer forces between the femoral and tibial joint surfaces by the development of circumferential (hoop) stresses.5 Furthermore, the viscoelastic behavior of meniscal fibrocartilage is essential for compressive load dissipation: the energy is absorbed by the compression of meniscal collagen fibers and the expulsion of the joint fluid through the tissue highly organized structure.6−11 Severe meniscal injuries are quite common and most often not possible to suture, and therefore treated by partial or total meniscectomy. This approach leads to most patients having an increased risk of developing symptomatic osteoarthritis and © 2018 American Chemical Society

Received: November 14, 2017 Accepted: March 15, 2018 Published: March 15, 2018 1518

DOI: 10.1021/acsbiomaterials.7b00879 ACS Biomater. Sci. Eng. 2018, 4, 1518−1527

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ACS Biomaterials Science & Engineering

The samples produced were labeled according to the PVA/porogen ratio and to the porogen provider, namely 1:5S, 1:7S, 1:8S, 1:9S, and 1:7F. Additional samples were produced without porogen agent and labeled as 0. 2.2. Porosity Measurement. The porosity of the hydrogels was evaluated by examining 10 μm thick cryotome slices of the hydrogels (n = 10) with a Leica optical microscope and the NIH ImageJ 1.49o software by applying the particle detection algorithm with a threshold of 100 μm2 and extracting the equivalent diameter. Slices were cut from different orthogonal planes to check morphological isotropy (data not shown) and pore distribution. Porosity, defined as the ratio between void and total volumes, and pore distribution corresponding to each different porogen amount were quantified, and the pore size values were converted from 2D to 3D through the scaling factor 1.273, as defined by the ASTM standard.36,37 2.3. Hydrogel Characterization. The water uptake and mass stability of the PVA hydrogels were evaluated by recording the mass variation of samples soaked in water over 65 days. The hydrogels were placed in deionized water at room temperature. The weight of each swollen hydrogel was measured at 0, 3, 7, 14, 21, 30, 45, and 65 days after removing external water excess by tapping the samples on absorbent paper; the bath water was changed after each measurement. The measured weight was compared to the initial mass to analyze the initial water-uptake and PVA weight stability in water. Thermogravimetric analysis (TGA) was performed on a Hi-Res TGA Q500 thermogravimetric analyzer (TA Instruments, New Castle, USA) under N2 atmosphere, to measure the total water content of the PVA hydrogels. Measurements were performed on 15−20 mg samples in an aluminum pan at a heating rate of 3 °C/min, from 30 to 600 °C. The weight loss (TG curve) and its first derivative (DTG curve) were recorded simultaneously as a function of temperature. 2.4. Mechanical Testing. The hydrogel specimens were cut in 10 mm side cuboids and tested in uniaxial unconfined compression with a Dynamic Mechanical Analyzer (DMA, Q800, TA Instruments, New Castle, USA) in a submersion fixture filled with phosphate buffered saline (PBS; Gibco, Grand Island, NY) at the controlled temperature of 37 °C. Stress relaxation tests were performed starting with a preload at 0.1% strain, followed by a 10%/min strain rate ramp until 10% compressive strain, which was held for 15 min; then another ramp was applied to reach 20% strain, and the deformation was held for another 15 min. The loading ramp was used to extract the compressive elastic modulus E; the relaxation portion of the stress relaxation curves was fitted using the software Origin Pro 8.6 with an exponential equation to extract the modulus at equilibrium Ha and the percentage of stress relaxation. At least 3 repetitions were performed for each material, data and errors are presented as average and standard deviation. 2.5. Meniscal Fibrochondrocytes Isolation. Visually intact adult bovine medial and lateral menisci (n = 20) were harvested from whole, intact knees (Research 87, Boylston, MA). From each knee, both menisci were dissected and immediately immersed in PBS supplemented with 5 mM ethylenediaminetetraacetic acid (EDTA; Sigma, St. Louis, MO), 0.5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma) and 1x Penicillin-Streptomycin (P/S; Gibco). EDTA, PMSF, and P/S were added for their metalloproteinase inhibition, serine protease inhibition, and antibacterial effect, respectively. The isolation protocol was optimized based on previously published protocols.38−40 Briefly, whole menisci were diced into 1 mm3 pieces and enzymatically digested with 1 mg/mL hyaluronidase for 1 h, 400 U/mL collagenase I for 6 to 8 h, 1000 U/mL collagenase II (Worthington biochemical Corporation, Lakewood, NJ) and 0.25% trypsin for 30 min. The primary cells found in solution after this sequence of treatments were washed, pooled, counted, and cryo-stored until use in the in vitro tests described below. 2.6. Cytotoxicity. To evaluate the potential cytotoxicity of the materials, primary meniscal fibrochondrocytes were seeded on the surface of the PVA porous hydrogels. The influence of different pores distribution on cellular activity was evaluated by testing samples made with both porogen agents. Cylinders of 5 mm diameter were obtained using sterile biopsy punches from ∼2 mm thick PVA hydrogel sheets. The samples were soaked for 48 h in Fetal Bovine Serum (FBS) to

a novel porous hydrogel-based implant for meniscal tear repair. A hydrogel-based material can approximate the high water content typical of native fibrocartilage. Furthermore, porosity in the submillimeter scale can potentially be incorporated within the bulk of the hydrogel to mimic the fluid exudation and pressurization of the natural tissue, also leading to tunable viscoelasticity to functionally mimic the load dissipation ability of meniscus. Porosity would also be beneficial to the integration of the implant with the native tissue, allowing cell migration19 and mechanical anchorage of the construct. To realize such an implant, we have used poly(vinyl alcohol) (PVA), a biocompatible material with long-term stability that is already known in the literature in multiple biomedical applications,20−24 including as meniscal implants.25−30 The potential of PVA hydrogel meniscus prostheses to improve histological scores in a rabbit meniscectomy model was demonstrated in vivo by Kobayashi et al.31 PVA hydrogels were utilized in sheep by Kelly et al.,32 but they found substantial failure due to the inability of the hydrogels to withstand the tensile hoop stresses experienced by the meniscus. As such, others have sought particles- or fiber-reinforced hydrogels.30,33,34 However, such solutions can present the typical drawbacks of composite materials: possible inhomogeneous mechanical and cellular response, and more complex processing, especially when producing a porous matrix, as in this case. Thus, the aim of this research was to design, develop, and validate a porous PVA hydrogel-based implant for meniscal repair that could be a possible surgical alternative to allograft implantations.

2. MATERIALS AND METHODS 2.1. Hydrogel Preparation. Research grade PVA (99+% hydrolyzed) with a molecular weight of 85−124 kDas and poly(vinylpyrrolidone) (PVP) with a molecular weight of 40 kDas were purchased from Sigma-Aldrich (St. Louis, MO) and used without further treatment. Polymer solutions were prepared by mixing 20% (w/v) of PVA and 4% PVP in deionized water. Addition of PVP was aimed at improving hydrogel network stability through interchain hydrogen bonding.29 The polymer solution was covered and placed in a humid environment at 120 °C for 90 min to ensure complete dissolution of PVA. The solution was cooled down to around 60 °C and mixed with a pore-forming agent in different PVA:porogen weight ratios (1:5, 1:7, 1:8, 1:9).20 The pore-forming agent was sodium hydrogen carbonate powder; to have two different granulometry distributions, sodium hydrogen carbonate powders from two different providers, namely Sigma-Aldrich S.r.l. (S5761) and Fisher Scientific (S233), were used. The differences in the particles sizes and distribution are highlighted from the porosity measurements, section 2.2. Physically cross-linked PVA-based hydrogels were prepared in water by the so-called “freeze−thaw” method, where microphase separation of PVA chains from water are achieved by taking the solution below the freezing temperature of water and thawing above freezing temperature of water in repeated cycles.35 Such procedure was chosen to avoid the use of toxic reagents during the manufacture. Here, PVA grafts were cross-linked by 5 cycles of 6 h freezing at −20 °C and 2 h thawing at room temperature. The PVA cryogels thus obtained were immersed in 0.1 N HCl solution at 50 °C and stirred for 48 h to remove sodium bicarbonate in the form of CO2 and achieve the desired porosity. The porous PVA hydrogels were washed several times in PBS buffer to stabilize the pH prior to any further investigation. Material optimization consisted of two steps: (1) the range of achievable porosity was characterized with PVA/porogen ratios of 1:5, 1:7, 1:8, and 1:9, manufactured with Sigma-Aldrich’s sodium bicarbonate through the salt-leaching technique; and (2) the difference arising from different granulometry, for a given PVA/ porogen ratio (1:7), was studied by comparing samples manufactured with either Sigma-Aldrich and Fisher porogen. 1519


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Figure 1. Production of porous PVA constructs and their morphological characterization: (a) Tunable porosity was achieved by mixing different amounts of porogen agents obtained from different sources (S, Sigma-Aldrich; F, Fisher): optical microscopy images for sections of different PVA:porogen ratios of 0, 1:5S, 1:8S, and 1:7F (scale bars 100 μm). (b) Measurement of porosity % according to the PVA/porogen ratio. * indicates a significant difference (p < 0.05). (c) Pore distribution in 1:7S and 1:7F, scaled for a 3D evaluation of pore diameters and normalized, showing that distribution was not affected by the PVA/porogen ratio and appeared constant across different samples, although it is possible to note that the F series induces larger pores for its coarser granulometry. performed in a manner similar to standard intraoperative procedure.42 The two edges of the samples were sutured to each other with 2 vertical loops 2 mm from the sample edge. Meniscus-meniscus and meniscus-PVA sutures strength was analyzed using an Instron 3365 Dual Column with a tension test pulling at 10 mm/min loading rate until failure. The ultimate failure loads (UTL) were extracted from the curves (n = 3). 2.8. Push-out Testing. Bovine menisci were dissected from the knee joints, and cylinders (8 mm diameter, 3 mm thick) were excised centrally in the axial direction (between the inner and outer part) using a dermal punch (Miltex, Plainsboro, NJ). To simulate a meniscus tear repair, a full thickness inner columnar defect (3 mm diameter) was carved with a dermal punch and then filled either with the same meniscus plug (sham control) or with a press fitted PVA porous hydrogel 4 mm diameter, 5−7 mm thickness plugs. The 1:7F PVA scaffold was chosen, as it presented the largest pores and highest pores interconnectivity. A group of samples was soaked for 48 h in FBS, and another group was treated for 2 h with a 0.1 mg/mL collagen I solution (Advanced BioMatrix Inc., CA) to favor cell adhesion and implant integration to the rest of meniscal tissue. Meniscus-PVA constructs were then cultured for 50 days in chondrogenic medium43 composed of DMEM supplemented with 1x PSF, 0.1 mM dexamethasone, 50 mg/mL ascorbate 2-phosphate, 40 mg/mL L-proline, 100 mg/mL sodium pyruvate, 1x insulin/transferrin/selenium (ITS) (Becton Dickinson, Franklin Lakes, NJ) with 10 ng/mL transforming

enhance cell initial adhesion. Each PVA cylinder was seeded in a nontissue culture treated 48 well plate with 5 × 103 primary meniscal bovine fibrochondrocytes suspended in 200 μL of DMEM containing 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin, and 25 μg/mL fungizone (PSF) (Invitrogen). A 200 μL volume of medium was used to avoid medium outflow from the scaffold and potential cell loss. After 2 h of incubation at 37 °C and 5% of CO2, each well was filled with medium to proceed with the culture time. At day 3 and 7, cell viability on the various PVA samples was evaluated using the Live/ Dead assay (Invitrogen). At day 3, 7, and 14, the metabolic activities of the cultures were measured using the MTS assay (CellTiter 96 Aqueous Cell Proliferation assay, Promega), as this provides a quantitative measurement related to the cell proliferation inferred from the Live/Dead assay. 2.7. Suture Strength Test. The robustness of the repair procedure, before cells colonization, was tested by suture strength measurements. Bovine meniscal tissues were obtained 24 h after slaughter and stored at −20 °C in PBS until testing. We assumed that one freezing-thawing cycle did not modify significantly the biomechanical properties of meniscal tissue.41 Menisci were excluded from testing if they were grossly degenerated or torn; planar samples of 15 × 10 mm and 2 mm thickness were obtained using a scalpel. Starting from the sample with the best ex vivo tests outcome, PVA samples of the same size were carved from 1:7F PVA slab in order to model a suture test. All suturing (Ethicon 2−0 Prolene) was 1520


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Figure 2. Hydrogel composition by thermal gravitometry and stability by water uptake/weight loss. (a) TGA analysis of PVA porous hydrogels used to quantify the weight loss occurring around 100 °C caused by water loss. (b) Water uptake of PVA hydrogels over 65 days. The recorded weight was normalized to the value at day 0 to analyze the initial water-uptake and the weight stability of PVA hydrogels in water. growth factor β3 (TGF-β3) (R&D Systems, Minneapolis, MN), which was renewed twice per week. Mechanical integration strength was evaluated at the beginning and at the end of culture using a custom testing device as previously described.44−46 Briefly, a Bose Electroforce mechanical tester was fitted with a 2 mm diameter flat-ended cylindrical indenter. This indenter was placed above a plate with a 5 mm diameter through-hole. The meniscus sample was placed on the plate, with the vertical axis of the defect aligned to the indenter. The indenter was then pressed through the defect site filling at a rate of 0.08 mm/s. The integration strength was calculated as the ratio of the maximum force recorded over the external surface between meniscus outer rim and PVA internal plug. Untested samples were also cryosectioned with a cryotome and analyzed histologically by Hematoxylin and Eosin staining, focusing on the profile of the PVA-meniscus junction. 2.9. Statistical Analysis. All results are presented as the mean ± Standard Deviation. Statistical significance was evaluated through oneway ANOVA, or unpaired t test where explicitly stated (significant value with p ≤ 0.05; n = 10 for porosity tests and n = 3 for pushout and MTS assays).

We compared the pore distribution of the 1:7 samples made with two porogens (Figure 1c): in both cases, the pore distribution is spread over a broad range, from 15 to 400 μm, with most of the pore diameters falling in the 20−100 μm range. The distribution for the F samples overlapped with that of the S samples but was slightly right shifted, indicating a higher number of larger pores, which is expected to enhance the fluid entrance and graft integration, as suggested by Vikingsson et al.48 and by Giannoni et al.49 3.2. Physicochemical Characterization. TGA measurements were performed on the nonporous PVA (0) and on the 1:5S, 1:7S, 1:8S and 1:7F samples to study the total amount of free water and its interaction with the PVA network. TGA curves (Figure 2a) show free water weight loss occurring around 100 °C. It was thus possible to quantify the free water percentage as a function of the porosity, starting from 75% for the nonporous PVA to 88% for the 1:8S sample. All the PVA hydrogels were stable up to 250 °C; the nonporous samples showed a decomposition temperature around 335 °C, whereas the decompositions of the 1:5 and the 1:8 samples started around 325 °C, similar to the values reported for equivalent PVA materials.50−52 PVA hydrogels water uptake and degradation were evaluated by recording the mass variation during 65 days of water immersion. The initial water uptake for the nonporous PVA was barely recordable (Figure 2b), whereas the porous hydrogels showed increasing values with decreasing of the PVA/porogen ratio; for the 1:7S and 1:8S samples, the water uptake was around 6 and 3% of the initial weight, respectively, after 7 days of swelling, whereas for the 1:5S ratio. the water uptake was 10% after 14 days. After the initial swelling, only small weight variations were detected, until the mass reached a stable value, after ca. 30 days. The plateau weight loss values of the 0, 1:5S, 1:7S, and 1:8S samples were 4.50, 1.40, 3.50, and 4.90%, respectively, of the maximum peak reached after the initial uptake. In other words, for porous scaffolds the weight loss increases with porosity, which can be explained by the associated increment of surface area. The low water intake was thus deemed suitable for the use of PVA hydrogels as medium term biomedical implants. Furthermore, the PVA stability in water suggested low degradation rate in physiological

3. RESULTS AND DISCUSSION 3.1. Hydrogel Preparation and Porosity. We prepared PVA hydrogels using a freeze−thawing method to cross-link the structure, to achieve the highest porogen miscibility within the PVA solution as reported by Shim.20 We were able to achieve slurries with good processability for PVA:porogen ratios in the range of 1:5 to 1:10 for the Sigma-Aldrich sodium bicarbonate, and only up to 1:7 for the Fisher sodium bicarbonate. Typical micrographs of slices from different samples are shown in Figure 1a, and the values of porosity calculated based on optical imaging are reported in Figure 1b. As expected, porosity increased with the total amount of porogen agent, with a significantly higher value for a given weight ratio, for the “F” samples (derived from Fisher sodium bicarbonate) compared to the “S” samples (derived from Sigma-Aldrich sodium bicarbonate). For the highest porogen content samples, namely 1:7F and 1:8S, we were able to reach 50% porosity, in the same range proposed by other authors for PVA porous hydrogels.35,47 Interestingly, no residual porogen was observed in any of the cut slices, suggesting a good interconnectivity of the porous network. 1521


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ACS Biomaterials Science & Engineering conditions, which would be ideal in a prosthetic scenario, as suggested by other authors.22,47 3.3. Mechanical Tests. The mechanical behavior of PVA hydrogels was evaluated by stress relaxation tests. Mechanical stiffness, measured as the compressive modulus E, was dependent on sample porosity and on the fluid pressurization state in the bulk, which was, in turn, a function of the permeability of the structure. The compressive modulus decreased with increasing PVA/porogen ratio, suggesting that the permeability to water is relatively low, so the trend is that of cellular solids: the higher the porosity, the lower the modulus. The steady viscoelastic response was evaluated through the aggregate modulus, i.e., the residual stress upon a given deformation at the steady state. The aggregate modulus values followed the same trend for all the “S” samples, with higher values for 20% strain and decreasing values as porosity increased. This suggested that greater porosity allowed the exudation of larger amounts of fluid from the construct. The 1:7F sample represents an outliner to both these trends, with higher values of both moduli than the rest of the porous materials, despite a similar porosity. This suggests that the different starting granulometry of the porogen agent affects the permeability of the constructs. The gap between the compressive modulus at 10% strain level and 20% is due to a pore-packing effect which increases the relative density. At 20% of strain, the compressive modulus varied from 350 kPa of the nonporous PVA hydrogel to 50 kPa of the 1:8 sample (Figure 3). Biomechanical studies of human menisci report a broad range of values, depending on testing parameters (stress state, strain rate5), location,53 and depth of the tested tissue,54 with aggregate moduli from 100 kPa to over 1 MPa, and Young’s moduli from about 500 kPa in compression to several MPa in tension.55 Compared to such reports, the values of our porous materials are placed in the bottom of the range, due to its porosity. Indeed, the nonporous samples have similar values of Young’s modulus as previous works.25,30 Clearly, the porous design entails a balance between cellular migration and stiffness to allow immediate loading of the joint. The initial disadvantage in terms of stiffness is expected to be compensated by the successive stronger integration with surrounding tissue, and by the reinforcement granted by new ingrowing tissue.19,56 3.4. Cytotoxicity. PVA porous hydrogels were seeded with bovine primary meniscal fibrochondrocytes to evaluate their cytotoxicity, following the procedure outlined in Figure 4a to minimize cell dispersion during seeding. Live/Dead assay showed the cell viability in cell-laden hydrogels (green marks viable cell; red, dead cells). Cell viability after 7 days remained high, in the case of the nonporous hydrogel as well as for both the porous structures of the representative “S” and “F” series that were chosen for this test (1:9S and 1:7F) (Figure 4b). Both porous scaffolds presented increased meniscal fibrochondrocytes viability, although cells generally maintained a roundlike shape, suggesting that the simple PVA mesh was not conducive to the promotion of cell adhesion. As expected, the presence of FBS significantly enhanced cell adhesion onto the scaffolds compared to the no FBS negative control (data not shown). The MTS assay results (Figure 4c) showed progressively increasing cellular metabolism during the 14-day culture in both cell-seeded, FBS treated porous samples, although only for the 1:9S sample increments are statistically significant. The nonporous sample, on the other hand, showed a significant drop after 7 days. Taken together, the Live/Dead and MTS

Figure 3. Mechanical characterization of PVA porous hydrogels. (a) Compressive modulus at 10% and 20% of strain, (b) aggregate equilibrium modulus, and (c) % of maximum stress peak relaxation.

results confirmed that the porous PVA hydrogels are cytocompatible, do not hinder cell metabolism, and are suitable for biomedical applications, as reported previously by other authors.57 3.5. Implant Integration. The results of the suture strength tests offer an insight on the behavior of the scaffolds in the period of time following surgical implantation. Of the pairings studied, the meniscus-meniscus coupling offered the highest UTL value, with the failure of the bond always localized on the suture (3 cases out of 3). The UTL value of the meniscus-PVA(1:7F) suture was 2.36 ± 0.13 N, lower than the value of meniscus-meniscus coupling of 9.17 ± 2.22 N, but within the same order of magnitude.58−60 This is likely due to the higher strength of the fibrous meniscus collagen structure compared to the PVA hydrogel. Although the lower resistance 1522


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Figure 4. In vitro test for PVA constructs biocompatibility: (a) General cell culture procedure for in vitro tests. After FBS adsorption and/or collagen surface coating, cells were let adhere to the upper surface of the hydrogel before medium addition for further culture. (b) Live/Dead analysis of cellladen PVA hydrogels. Primary meniscal fibrochondrocytes were isolated from bovine menisci and cultured on PVA porous hydrogels. Fluorescence images were taken at different time-points, scale bar 100 μm. (c) MTS assay of cell-laden hydrogels after treatment with FBS and further culturing. The nonporous samples showed a reduction in cell metabolism, suggesting a less suitable environment for cell adhesion and proliferation, whereas the porous samples showed increasing metabolic states as a function of culture times. * indicate a significant difference (p < 0.05).

already at day 0. The pushout force required to remove the inner PVA plug from the outer rim of the native meniscus at the end of the culture (day 50), shown in Figure 5d, was slightly higher than at day 0, albeit with no statistically significant difference, suggesting moderate time-dependent meniscal tissue ingrowth into the cavities offered by the PVA structure. The samples treated with FBS and collagen type I showed the highest degree of integration after 50 days, potentially stronger than the simple sham control group, in the pushout test. On the basis of histological sections taken from these samples (Figure 6), the PVA hydrogels were found to fit and match the inner rim of the native tissue defect after 50 days in culture. At day 0, however, histological processing caused moderate shrinking of the PVA scaffold. The integration with the surrounding meniscal tissue at day 50 was sufficient to avoid this artifact. In the sham control group, fracture fibers from the biopsy punch remained visible at the end of the culture in the internal part of the sample, while some repair did

of PVA compared to native meniscus could suggest suturing together the torn pieces of a meniscus as a preferred option from a clinical perspective, the torn meniscal portion is often damaged and not a suitable match for its original location. Thus, PVA hydrogel that could be shaped appropriately could be a suitable surgical alternative to restore biomechanical functions with suture strength in the order of native tissue control. The implant integration was assessed through a pushout test, in which we compared the maximum force at day 0 and day 50 for a sham of native tissue, the porous 1:7S scaffold pretreated in FBS, and the same scaffold type pretreated with FBS and a collagen solution (Figure 5). The sham test had lower integration strength at day 0, when the native tissue just punched out did not tightly fit into the defect, as confirmed by histology (Figure 6a). By day 50, sham native tissue integration strength improved significantly, as reported in Figure 5d. The PVA scaffold adapted well to the defect site and fit tightly, resulting in higher forces required to dislodge the scaffold 1523


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Figure 5. Implant integration analyzed by mechanical testing: (a) sketch of the ex vivo plug dimensions. (b) Picture of the plug at the beginning of the culture where the interface between the native outer rim and the PVA implant placed in the core is visible. (c) Push out experimental platform. (d) Data from the analysis of integration strength of different samples combinations measured at day 0 and day 50. * indicates a significant difference (p < 0.05).

Figure 6. Ex vivo analysis of implant integration: day 0 histology (H&E) for (a) the sham and (b) the 1:7F samples. The clear gap between the native tissue and the PVA edge was clearly visible. After 50 days in culture, remodeling of the native tissue was seen (c) in the sham sample, revealing the presence of an internal crack under the surface, which showed integration due to migration cells on the surface of the explant, whereas the crack or gap was absent in the explants fitted with a PVA implant, treated with (d) FBS only, or with (e) FBS and collagen type I. Scale bar = 1 mm.

occur at the outer surface of the defect site, likely due to migration of cells located on the outer surface (Figure 6c, and inset). For the PVA implants, the gap at day 50 was reduced to a minimum width, and the porosity of the PVA was preserved

(Figure 6d, e). The fact that our constructs reached the same integration strength as native tissue controls confirms their relatively good integration, likely more effective in this specific respect than other reported results;61 on the other hand, poor 1524


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for its support. This work is supported in part by the U.S. Department of Defense (DOD W81XWH-15-1-0104) (R.S.T).

cell migration to the PVA implant is a limitation of this study and was likely a consequence of the static culture approach adopted in this characterization. In fact, without mechanical stimulation, diffusion of nutrients to the deep regions of the meniscal plug is hindered by the dense extracellular matrix.

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4. CONCLUSIONS PVA hydrogel represents a promising material for biomedical applications in view of its tissue-like mechanical behavior and biocompatibility, with the potential to replace rigid prosthesis components and serve as a synthetic meniscal cartilage-like structure. In this work, we were able to design and develop a porous PVA hydrogel implant for meniscus fibrocartilage with an open porosity that enhances synovial fluid exudation during mechanical stresses and improves implant integration, while preserving mechanical properties close to those of native tissue. With our approach, the specific balance between the elastic requirements of native meniscus and improved integration can be approached by appropriate optimization of the porosity of the graft. Our results showed that the viscoelasticity of PVA hydrogels is comparable to that of the native biological tissue. An evident stress-relaxation behavior confirms that the porous structure is able, after a compressive action, to exudate the internal aqueous fluid phase with a specific time-dependency as a function of its relative porosity. To assess the potential use of PVA hydrogel as a meniscal graft, were carried out tests under ex vivo culture conditions and by means of suture test analyzing the interaction of PVA with native meniscal fibrocartilage. Ex vivo culture identified PVA as an inert material for its reaction to the native cartilaginous tissue, which is a key parameter for prosthesis integration, although in vivo studies will be required to assess any broader host response interaction. The porosity, on the other hand, offers the possibility for good interplay between tissue and implant, with a good tissue penetration along the rough external surface of the PVA implant. Mechanical testing values from the suture tests showed lower interconnectivity but within the same order of magnitude between PVA/meniscus and meniscus/meniscus. Thus, PVA-based constructs presented here were found to be stable hydrogels, displaying mechanical and morphological characteristics similar to those of native meniscus, with good biocompatibility, making this porous hydrogel a promising option for functional repair of the meniscal tissue.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +39 010 71781241. ORCID

Luca Ceseracciu: 0000-0003-3296-8051 Author Contributions †

L.C. and R.G. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Luigi Molfetta for kindly assisting with the implant integration analyses and Lara Marini for the PVA characterization. R.G. acknowledges the Ri.MED Foundation 1525


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Porous Poly(vinyl alcohol)-Based Hydrogel for Knee Meniscus

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