PVA-β-TCP bilayered hydrogels for articular cartilage tissue repair

Accepted Manuscript Preparation and characterization of PVA-PEEK/PVA-β-TCP bilayered hydrogels for articular cartilage tissue repair Weichang Li, Junpei Kang, Yulin Yuan, Fengcai Xiao, Hang Yao, Sa Liu, Jianxi Lu, Yingjun Wang, Zhen Wang, Li Ren PII:

S0266-3538(16)30100-2

DOI:

10.1016/j.compscitech.2016.03.013

Reference:

CSTE 6361

To appear in:

Composites Science and Technology

Received Date: 27 December 2015 Revised Date:

6 March 2016

Accepted Date: 12 March 2016

Please cite this article as: Li W, Kang J, Yuan Y, Xiao F, Yao H, Liu S, Lu J, Wang Y, Wang Z, Ren L, Preparation and characterization of PVA-PEEK/PVA-β-TCP bilayered hydrogels for articular cartilage tissue repair, Composites Science and Technology (2016), doi: 10.1016/j.compscitech.2016.03.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Preparation and characterization of PVA-PEEK/PVA-β-TCP bilayered hydrogels for articular cartilage tissue repair Weichang Lia*, Junpei Kanga*, Yulin Yuanb, Fengcai Xiaoa, Hang Yaoa, Sa Liua, Jianxi

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Luc, Yingjun Wanga, Zhen Wangb**, Li Rena**

Technology, Guangzhou 510641, China

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a． School of Materials Science and Engineering, South China University of

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b． Department of Orthopaedic Oncology, Xijing Hospital, the Fourth Military Medical University，Xi’an 710032, China

c． Shanghai Bio-Lu Biomaterials Co. Ltd., Shanghai 200335, China

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*These authors contributed equally to this work.

* *Corresponding authors. E-mail addresses: [email protected] (Zhen Wang), [email protected] (Li Ren)

ACCEPTED MANUSCRIPT Abstract: The poly(vinyl alcohol)(PVA) hydrogel is regarded as a potential articular cartilage replacement for its good biocompatibility, high permeability to fluid and load-bearing This

work

investigated

alcohol)-polyetheretherketone/Poly(vinyl

a

novel

Poly(vinyl

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properties.

alcohol)-β-tricalcium

phosphate

(PVA-PEEK/PVA-β-TCP) bilayered hydrogels by freezing-thawing with biomimetic

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properties for articular cartilage and subchondral bone is developed. The bilayered

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hydrogels microarchitecture consists of a highly porous and dense structure, and the internal structure were analyzed by micro-CT. The morphology of the resulting hydrogels was analyzed by scanning electron microscopy (SEM), the enhancement of the mechanical properties of the PVA-PEEK/PVA-β-TCP bilayered hydrogels were

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demonstrated by mechanical testing. The bilayered structure indicate that a good bonding exist between the two layers, which is known to be a requisite necessary to assure a good integrity and functionality of the osteochondral construct. In addition, in

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vitro cell culture studies revealed that the hydrogels has no negative effect on the cell

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viability and proliferation and possess good biocompatibility. Then, the bilayered hydrogels were implanted into the knee joint defect of rabbits and hematoxylin and eosin and immunohistochemical staining. The PVA-PEEK/PVA-β-TCP bilayered hydrogels show good potential for use in the field of articular cartilage repair. Key Words: poly(vinyl alcohol)(PVA), Articular cartilage, PVA-PEEK/PVA-β-TCP bilayered hydrogels, Microstructure

ACCEPTED MANUSCRIPT 1. Introduction Articular cartilage is as a soft conjoin cushion between bones in arthrosis which providing the tissue with its unique properties, and have been playing an important

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role in surface lubricative and biomechanical properties, which in order to reduce and transfer stress and abrasion in arthrosis [1, 2]. Articular cartilage defects occur frequently as a result of sports related trauma or in osteoarthritis, congenital defects,

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leading to pains or dysfunctions and other causes of loss of skeletal tissue. Various

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therapeutic options for an osteochondral defect have been taken to reproduce the tissue function, such as conservative measures, osteochondral transplantation and autologous chondrocyte implantation. However, there appears to be other deficiencies as well. All of the current procedures are collecting autologous grafts damages the

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healthy body and the amount that can be obtained is limited [3]. A growing emphasis on synthetic polymers has led to the study of hydrogels as materials with promising treatment for substitute or regeneration of damaged articular cartilage [4]. The

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application of hydrogels in broad range of domains leads to the manipulation of their

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physical properties. Since the hydrogels are hydrophilic, cross-linked network of polymers, they are usually biocompatible in nature and are nonirritating to the articular cartilage tissues when in contact with them [5-7]. Hydrogels based on multitudinous polymers have been studied for replacement or

repair of damaged articular cartilage including poly(lactic-co-glycolic acid) (PLGA) [8],

hyaluronic

acid/poly(ethylene

glycol)

(HA/PEG)

[9],

polyvinyl

alcohol/poly(vinyl pyrrolidone) (PVA/PVP) [10], collagen and gelatin [11]. One of the

ACCEPTED MANUSCRIPT most extensively researched hydrogels for the cartilage replacement is polyvinyl alcohol (PVA) [12], hydrogels based on PVA have been prepared with similar swelling, load-bearing properties, excellent biocompatibility and fluid flow properties

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to native cartilage [13]. Nevertheless, mechanical strength and lubrication of PVA hydrogels still cannot meet the demand of natural cartilage due to the strong action of the hydrogen bond formed in inner molecules [10]. In order to improve the

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mechanical properties and the surface lubrication, polyetheretherketone (PEEK),

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which is an easy processing thermoplastic polymer and has been used for many biomedical applications due to its high elastic modulus, thermal stability and chemical resistance [14]. In recent years, many researches have been interested in improving wear resistance and decreasing friction coefficient of PEEK composites [15-17]. In

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this article, PEEK is selected as the filler of PVA-based hydrogels owing to its outstanding merits of high mechanical strength, elastic modulus, and thermal stability. Articular cartilage defects often occurs with subchondral bone regions cartilage

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injuries. There are different defects of articular cartilage with respect to the depth, size,

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and lesion locale including matrix disruption, partial-thickness and full-thickness [18]. The substitute or repair material of PVA-based hydrogels does not meet the need for treatment. Hence, designing and preparation of this tissue have remained challenging. One of the most promising strategies consists of the generation of a bilayered structure, obtained by the combination of distinct but integrated layers corresponding to the cartilage and subchondral bone regions. Such design is because the bilayered construct of the subchondral bone regions and the cartilage layer of grows under

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different

biological,

biochemical,

and

biomechanical

properties

requirements, which is based on previously reported study [19]. The lower of bilayered

material

comprised

of

polyvinyl

alcohol/β-tricalcium

phosphate

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(PVA/β-TCP) could be achieved by preparation of hydrogels contain inter-connected porous network. The combination of β-TCP with PVA can enhance not only mechanical properties and bioactivity but also adherent to around natural tissue, and

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promotes cell adhesion and proliferation. The intermediate layer between the two

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layers that could be useful for increasing the bonding of the two layers and also could be helpful in promoting differential growth of the osteochondral constructs [19]. In this study, we address some of the most common natural and synthetic polymers including strategies for fabrication of blends and composite bilayered

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hydrogels. To exploit the advantages of among PEEK, β-TCP and PVA, we fabricated a PVA-PEEK/PVA-β-TCP bilayered hydrogels through freezing and thawing methods. In these systems, PEEK defines a support solid structure contributing to rigidity and

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mechanical resistance [20], while the second compound, β-TCP, have proven to be

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osteoinductive and osteoconductive [21,22]. The morphology, mechanical properties and moisture content of the PVA-PEEK/PVA-β-TCP bilayered hydrogel were examined. In vitro and in vivo experiments were performed to assess the biocompatibility and the effect of repair. The resulting bilayered hydrogel exhibited a good biocompatibility and can achieve satisfactory effect.

2. Materials and methods

ACCEPTED MANUSCRIPT 2.1. Materials PVA (saponified greater than 99%, Mw=89,000–98,000) was purchased from Sigma-Aldrich (St. Louis, MO). Medical grade semicrystalline PEEK was obtained

2.2 Preparation of bilayered hydrogels

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from Victrex Co., UK. β-TCP was procured from Aladdin Reagent Co., Ltd. (China).

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PVA composite hydrogels were prepared by a freezing-thawing method [23].

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Briefly, The PVA solution and PEEK solution were prepared by dissolving them in dimethyl sulfoxide at 85oC with stirring for 6h, respectively. Then, a mixtures are obtained by mixing the two solutions and stirring in round-bottomed flask for at least 24 h to facilitate dispersion of PVA and PEEK. And, the PVA content was adjusted to

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75 wt % of the PEEK mass. The mixture of PVA-β-TCP can also be prepared by the same method. After held at vacuum to remove air bubbles, the mixtures of PVA-PEEK and PVA-β-TCP were poured into a self-made mould and with

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temperature set at -20oC, was placed in a refrigerator for 20 h. Then it was taken out

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and thawed at room temperature for 6 h. The freezing and thawing process was repeated 9 times. The PVA-PEEK/PVA-β-TCP bilayered hydrogels (A-K/A-P) so obtained were washed thoroughly to wash off the residual solvents. As control group, the PVA-PEEK hydrogels (A-K), PVA-β-TCP hydrogels (A-P) and PVA/PVA (A/A) bilayered hydrogels were prepared in the same way.

2.3 Characterization

ACCEPTED MANUSCRIPT A small cut-offs from the sample were scanned using Skyscan 1172 micro-CT system (Bruker, Billerica, MA), at image a pixel size of 8.1 µm and 1 mm Al Filter, with a X-ray source voltage and current of 80 kV and 110 µA. Following scanning,

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three dimensional (3-D) reconstructions were performed by an application of a global threshold and the projected files were reconstructed using the Nrecon V1.4 (Bruker) cone beam reconstruction program. The morphologies of the hydrogels was also

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observed with a Philips XL-30 environment scanning electron microscope at 20 kV.

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The A-K and A-P were also studied as the blank sample. To maintain the real microstructure of the hydrogel under wet conditions, all of the samples were observed directly without any pretreatment. Fourier transform infrared ray (FTIR, Thermo-Nicolet, USA) spectroscopy was performed in the range of 4000 to 500 cm−1

2.4 Swelling analysis

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with the in order to evaluate the bonding between polymers of the blend hydrogels.

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To estimate the swelling behavior of the hydrogels, the samples were incubated in

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PBS buffer (pH 7.4) at 37 oC. At select time points, the mass of the samples was weighed periodically until the swelling behavior of the hydrogels reached the equilibrium stage. The swollen hydrogels were weighed after the excess of water on the surface was absorbed with filter paper. The swelling ratio (SR) of the samples was then calculated as follows: SR = (Ws–Wd)/Wd. where Ws and Wd are the weights of the hydrogels at the swelling state and at the dry state, respectively.

ACCEPTED MANUSCRIPT 2.5 Mechanical properties The mechanical properties were carried out on a mechanical testing machine (BOSE ElectroForece3200, USA) at a rate of 0.5 mm/min. The swollen hydrogel

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samples were manufactured as a shape of cylinders with 10 mm in diameter and 5 mm in height. The samples were sandwiched between a porous, rigid barrier and was constrained laterally in order to prevent outward expansion during the uniaxial

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compression. The porous barrier was in contact with an external reservoir containing

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deionized water, permitting fluid flow between the sample and bath. The dynamic mechanical behavior of the hydrogels was evaluated using the same mechanical testing machine. Standardized strip specimens (2mm × 5mm) were cut from the hydrogels and stretched at room temperature with a constant speed of 10 mm/min. At

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least five replicates of each type of specimen were tested.

2.6 Cell viability assay

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To explore the cytotoxicity of the PVA hydrogels, L929 cells were treated with the

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hydrogels, and cell viability was determined using the methylthiazolyl tetrazolium (MTT) assay. L929 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)

medium

with

10%

fetal

bovine

serum

(FBS)

and

1%

penicillin-streptomycin, and maintained at 37 oC in a 5% CO2 incubator. The culture medium was changed every 2 days. The sterilized hydrogels were next immersed in culture medium at 37 oC in a 5% CO2 atmosphere for 24 h, and then the leaching liquor was used as the L929 cells growth medium. Viability of the seeded L929 cells

ACCEPTED MANUSCRIPT was studied by MTT assay. The optical density of the solutions was recorded using a microplate reader at a wavelength of 570 nm.

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2.7 Animal Surgical procedure The animal experiments conducted in this study were authorized by the Animal Ethics Committee of Sun Yat-sen University and was carried out in accordance with

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guidelines for the care and use of laboratory animals. During surgery, four female

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rabbits were used and general anesthetized. For the insertion of the implants, the rabbits were initially immobilized and placed in a supine position. A lateral parapatellar longitudinal incision was made to expose the knee joint. With the knee maximally flexed, a defect was created in the center of the groove. Then, the debris

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were removed from the defect with a curette and rinse. The A-K/A-P (It used was cylindrical with a diameter of 4 mm and height of 10 mm) implants were placed in the defect, and the muscle and skin were closed with sutures (Fig. 6). The rabbits were

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sacrificed at 2, 4, 8 and 14 weeks after surgery.

2.8 Histological observation For

histopathological

analysis,

condyles

were

fixed

in

4

%

(v/v)

paraformaldehyde at room temperature, decalcified with 5 % (v/v) formic acid for 4-6 weeks and embedded in paraffin wax. The samples were sectioned; representative sections were cut in the mesiodistal plane and stained with hematoxylin and eosin (H&E) and examined by light microscopy

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3. Results and discussion 3.1 Morphology of hydrogels

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Fig. 1a shows the macroscopic appearance of a pure PVA hydrogels and related composite hydrogels. The micro-CT techniques can show detail down to around a micron scale and is widely used for characterization of internal geometry of hydrogels.

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We applied micro-CT to the observation of internal geometry of 3D reconstructions of

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A-K/A-P. Form fig. 1b-1d, we observed that the microarchitecture consists of a highly porous and dense structure with characteristics similar to natural articular cartilage. As osteochondral transplantation are used in a physiological environment, hydrogels studied in wet conditions could reflect their true microstructure. Thus, the samples in

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this study were observed in wet conditions by environment scanning electron microscopy (ESEM). The bilayered hydrogels’ microarchitecture consists of a highly porous and dense structure (Fig. 2c-2f). Fig. 2c and 2e demonstrates the

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microstructure of the pure PVA bilayered hydrogels. The surface structure of the

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hydrogels were homogeneous, and there were no obvious particle on the surface. The typical micrographs of the cross-sections of A-K/A-P bilayered hydrogel showed etched features and rough structures with many granule present on the surface (Fig. 2d). Furthermore, the lower layer of porous have an internal three-dimensional porous structure with lots of micropores on the surface, and pore sizes recommended for cartilage tissue engineering scaffolds are 100-200 µm, with a desired porosity of ~90% to provide sufficient space and surface area for cell seeding in the temporary

ACCEPTED MANUSCRIPT hydrogel prior to implantation [19, 24]. These structures indicate that a good bonding exist between the two layers, which is known to be a requisite necessary to assure a good integrity and functionality of the osteochondral construct. The lower layer of

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porous pure PVA bilayered hydrogels had showed a smooth porous network structure, whereas the A-K/A-P was a relatively homogeneous and porous structure with good pore connectivity. The structures of A-K and A-P were not significantly different from

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the composite bilayered hydrogel, and the formation of the bilayered structure did not

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affect the pore size, the porosity and the microstructures.

Fig. 3 shows the infrared spectrum analysis of samples with freeze-dried. It can be observed that the characteristic adsorption peaks at 3247 cm−1 and 1074 cm−1 represent -OH and -CO- vibration of PVA. FTIR spectrum of A-P hydrogels shows the

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characteristic adsorption peaks in PVA and β-TCP quite well. The spectra peaks around 1016 cm-1 and 1425 cm–1 indicating the presence of PO43- and CO32- in β-TCP. The most drastic change of FTIR spectra of the A-K is the appearance of some strong

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absorption peaks around 1004 cm-1 and 1650 cm–1, which indicates the presence of

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ether bond and carbonyl group of PEEK. Compared with the spectra of A-K, the absorption peaks at 1074 cm−1 of PVA shifts higher, which may be attributed to strong interactions between the hydroxyl groups of PVA and the carboxyl groups of PEEK.

3.2 Swelling properties Swelling properties of the hydrogels are important parameter for substance exchange when used for biomedical applications. Many properties of hydrogels, such

ACCEPTED MANUSCRIPT as flexibility and mechanical strength, are mainly related to the swelling ratios [9, 25]. The influences of filling polymerization and morphological structure on the swelling ratios of PVA composite hydrogels immersed in PBS are shown in Fig. 4. All the

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samples were reached equilibrium within 10 h, indicating that hydrogels have fast swelling characteristics. There is signicant difference between porous A-P and others due to its largely dependent on the property to maintain the porous structure, suitable

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for water absorption. The swelling ratios of the composite hydrogels were dependent

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on the hydrophilicity of the materials [26]. PVA is a highly hydrophilic material because of its chemical and physical structure. The swelling properties of dense structure mainly depended on the hydrophilic ability of the functional groups and the effective cross-link density of the hydrogels, and the swelling ratio of the hydrogels of

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dense structure decreases significantly due to the denser inner structure. The swelling ratios of A-K/A-P bilayered hydrogels is larger than A/A due to inclusion of the composite fillers of PEEK and β-TCP, indicating that the addition of that

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reinforcement influences the crosslinking of the matrix. The swelling ratio in

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crosslinked samples is desirable since it is comparable to the natural articular cartilage in which water is one of its major constituents [27].

3.3 Mechanical properties Mechanical properties of the replacement are very important to minimize interfacial strain mismatch at the implant/tissue interface [28]. Hydrogels were tested for their mechanical properties under compression. Fig. 5a reveals the resulting

ACCEPTED MANUSCRIPT compressive stress-strain characteristics of hydrogels. The mechanical properties of dense structure were improved significantly when compared with the hydrogels of porous structure. We observed that the structure and mole ratio of composite fillers

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had a big effect on the stress-strain behavior. Reinforcement with PEEK and β-TCP clearly improved mechanical strength over demonstrated pure PVA hydrogels. The interaction between the PVA matrix and the fillers of PEEK and β-TCP may have

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been augmented through crosslinking, which permitted stress transfer at the interface

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between the PVA and the fillers of PEEK or β-TCP. Thus, the mechanical properties of the A-K/A-P bilayered hydrogels depended not only on the structure of composite, but also on the reinforcing behavior of the fillers. Although the compressive properties were worse than those of the pure PVA hydrogels of dense structure, the

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values were in the acceptable range (many tissue engineering hydrogel for articular cartilage repair present poor compressive properties [29, 30]). Furthermore, the mechanical properties of the hydrogels were investigated by the tensile test. It can be

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observed that the hydrogels had showed satisfactory mechanical properties, especially

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the A-K/A-P bilayered hydrogels (Fig. 5). With the adding of PEEK and β-TCP, the tensile strength of the A-K/A-P was enhanced significantly when compared with those of the pure A/A bilayered hydrogels. In summary, in this work, we demonstrated that fillers are useful for enhancing the stability of composite hydrogels, which depending on the nature of the host matrix and matrix/filler interactions.

3.4 Cytocompatibility studies

ACCEPTED MANUSCRIPT Fig. 6 exhibition the results of the cytocompatibility for all cells in comparison with the control, with the culture time elongation, all groups displayed an increase in MTT values. No difference was observed among the A-K, A-P, A/A and A-K/A-P

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groups during the study. The blank control groups were cells without hydrogels. These results demonstrate that all samples have cell viability more than 90%. The

3.5 Histological examinations

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hydrogels exhibited obvious cytotoxicity.

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results illustrated that all of the hydrogels were biocompatible and none of the

To evaluate the effect of repair and replacement, at 2, 4, 8 and 14 weeks after surgical, the rabbits were sacrificed for histological assessments. The histological

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findings according to H&E staining are shown in Fig. 8. H&E staining of sections of implanted A-K/A-P bilayered hydrogels after 2 weeks and a mild infiltration of inflammatory cells and fibroblasts (Fig. 8a). After 4 weeks, Fig. 8b showed

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compatibility with surrounding tissue and maintained ideal architecture without local

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inflammation. After 8 weeks, the implanted hydrogel could be distinguished from the host tissues becoming confluent and mature (Fig. 8c) and newly generated cartilaginous formation appeared in a closely knitted arrangement throughout the implanted constructs after 12 weeks (Fig. 8d). The results of the bilayered structures seem more advantageous for osteochondral tissue engineering applications for a number of fundamental reasons

ACCEPTED MANUSCRIPT Conclusions In this study, A-K/A-P bilayer hydrogel was fabricated by freezing-thawing cycle’s method. The results demonstrate that bilayer hydrogel microarchitecture

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consisted of a highly porous and dense structure respectively. With the additional of fillers of PEEK and β-TCP, the microstructure of the hydrogels becomes etched features and rough structures, which leads to the decrease of the swelling ratio and

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mechanical properties. The in vitro cell culture results showed that the composite

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hydrogels had excellent biocompatibility. In light of animal experiments and histological observation, though further understanding of the mechanisms involved in refill tissue formation for articular cartilage repair is necessary, it can be concluded

Acknowledgements

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that A-K/A-P is a potential articular cartilage repair material.

This work was supported by Science and Technology Innovation Council of

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Guangzhou, China (201508020123), National Natural Science Foundation of China

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(51273072), National Nature Science Foundation of China (Grants 51232002) and National Basic Research Project of China (2012BAI17B02).

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Fig.1. Macrostructure (a) and 3D micro-CT-reconstructed images (b) of A-K/A-P, and the microarchitecture consists of dense structure (c) and highly porous (d).

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Fig 2. SEM images of surfaces for A-K layer (a) and A-P porous layer (b), fracture

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surfaces for bilayered A/A (c, e) and A-K/A-P(d, f).

PVA

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A-P

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A-K

4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1) Fig 3. FTIR spectrum of the hydrogels of pure PVA, A-P and A-K.

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30

15

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Swelling ratio

45

0

A-K

A-P

A/A

A-K/A-P

TE D

M AN U

SC

Fig. 4. Equilibrium swelling ratio of the hydrogels at 37 oC.

Absorbance (A570)

AC C

EP

Fig. 5. Typical compressive (a) and tensile (b) stress-strain curves for the hydrogels.

1.5

A-K A-P A/A A-K/A-P contral

1.0

0.5

0.0 Day 1

Day 3

Day 5

Day 7

Time

Fig. 6. L929 cell viability with different hydrogels as a function of culture time.

A-K/A-P bilayered hydrogel cylinder implant into the defect in the center of

EP

TE D

M AN U

the knee.

SC

Fig. 7.

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AC C

Fig. 8. H&E staining of implanted bilayered hydrogel of A-K/A-P for 2 (a), 4 (b), 8 (c) and 14 (d) weeks after surgical.