A Porous Polyvinyl Alcohol-based Hydrogel for Knee Meniscus

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Characterization, Synthesis, and Modifications

A Porous Polyvinyl Alcohol-based Hydrogel for Knee Meniscus Functional Repair Luca Coluccino, Riccardo Gottardi, Farouk Ayadi, Athanassia Athanassiou, Rocky S Tuan, and Luca Ceseracciu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00879 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 17, 2018

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ACS Biomaterials Science & Engineering

A Porous Polyvinyl Alcohol-based Hydrogel for Knee Meniscus Functional Repair Luca Coluccinoa,b,‡, Riccardo Gottardib,c,‡, Farouk Ayadid, Athanassia Athanassioua, Rocky S. Tuanb, Luca Ceseracciue,* a

Smart Materials, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy

b

Department of Orthopaedic Surgery, Department of Chemical Engineering, and the McGowan

Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA c

Ri.MED Foundation, Palermo, Italy

d

Normandie Univ, UNIROUEN, INSA Rouen, CNRS, PBS, 76000 Rouen, France

e

Materials Characterization Facility, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa,

Italy ‡These authors contributed equally. AUTHOR INFORMATION Corresponding Author * Luca Ceseracciu Materials Characterization Facility Istituto Italiano di Tecnologia Via Morego 30 16163 Genova, Italy

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Phone office +39 010 71781241 Email [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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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 polyvinyl 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

The meniscus fibrocartilage plays a key role in knee joint biomechanics in terms of load transmission, shock absorption, and general proprioception1–4. The menisci mainly transfer forces between the femoral and tibial joint surfaces by the development of circumferential (hoop) stresses5. Furthermore,



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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 structure6–11. 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 other chronic, degenerative joint diseases. The restoration of meniscal functions with a bio-stable 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 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 sub-millimeter 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 polyvinylalcohol (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 ACS Paragon Plus Environment

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hydrogels30,33,34. 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

2.1

MATERIALS AND METHODS Hydrogel preparation

Research grade PVA (99+% hydrolyzed) with a molecular weight of 85-124 kilodaltons and poly(vinyl pyrrolidone) (PVP) with a molecular weight of 40 kilodaltons 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 minutes 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 crosslinked 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 crosslinked by 5 cycles of 6 hour ACS Paragon Plus Environment


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freezing at -20 °C and 2 hour thawing at room temperature. The PVA cryogels thus obtained were immersed in 0.1 N HCl solution at 50 °C and stirred for 48 hours 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. 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 labelled 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 standard36,37. 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 ACS Paragon Plus Environment

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room temperature. The weight of each swollen hydrogel was measured at 0, 3, 7, 14, 21, 30, 45, 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 minutes; then another ramp was applied to reach 20% strain, and the deformation was held for another 15 minutes. 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 5mM 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 anti-bacterial 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 hour, 400 U/mL collagenase I for 6 to 8 hours, 1000 U/mL collagenase II (Worthington biochemical Corporation, Lakewood, NJ) and 0.25% trypsin for 30 minutes. 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 hours in Fetal Bovine Serum (FBS) to enhance cell initial adhesion. Each PVA cylinder was seeded in a non-tissue culture treated 48 well plate with 5 x 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 hours 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


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(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 hours 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 tissue41. Menisci were excluded from testing if they were grossly degenerated or torn; planar samples of 15 x 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 performed in a manner similar to standard intraoperative procedure42. 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 (8mm diameter, 3mm 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



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PVA scaffold was chosen, as it presented the largest pores and highest pores interconnectivity. A group of samples was soaked for 48 hours in FBS, and another group was treated for 2 hours 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 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 described44–46. 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 one-way 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). 3

RESULTS AND DISCUSSION


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3.1

Hydrogel preparation and porosity

We prepared PVA hydrogels using a freeze-thawing method to crosslink the structure, to achieve the highest porogen miscibility within the PVA solution as reported by Shim20. We were able to achieve slurries with good processibility for PVA:porogen ratios in the range of 1:5 to 1:10 for the SigmaAldrich sodium bicarbonate, and only up to 1:7 for the Fisher sodium bicarbonate. Typical micrographs of slices from different samples are shown in Fig. 1a, and the values of porosity calculated based on optical imaging are reported in Fig. 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 SigmaAldrich 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. We compared the pore distribution of the 1:7 samples made with two porogens (fig. 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.



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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 - 0, 1:5S, 1:8S, 1:7F (Scale bars 100 µm). (b) Measurement of porosity % according to the PVA/porogen ratio. * indicate a significant difference (p

A Porous Polyvinyl Alcohol-based Hydrogel for Knee Meniscus

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