1. Introduction
Bone transplantation is the second most common type of tissue transplantation following blood transfusion [
1]. Bone grafting is considered as a well-described technique of bone defect treatment [
2]. Bone grafting refers to the transplantation of an autograph or an allograft. Despite both transplantation types meeting the requirements of appropriate properties (osteoconductivity, osteoinductivity, osteointegration), there are many disadvantages, e.g., low availability of grafts, blood loss, longer surgical time, infection etc. Bone tissue engineering tries to eliminate these problems by the application of fully biocompatible scaffolds based on natural or synthetic materials serving as matrices for cell incorporation and cultivation support for renewal of healthy tissue [
1,
2].
The main objective of tissue engineering is to restore and improve the function of tissues by preparing porous three-dimensional (3D) scaffolds, and seeding them with cells and growth factors [
3]. The term scaffold is used for 3D biomaterial that provides a suitable environment to promote cell proliferation, osteogenic differentiation, and production of extracellular matrix (ECM) in order to regenerate tissues and organs. Currently, there is the aim to produce scaffolds able to provide regenerative signals to cells [
4]. For this purpose, efforts are being made to develop scaffolds based on biomaterials that mimic those found in the natural environment [
1]. Attention must be paid to the design and composition of the desired scaffolds [
5]. Different types of materials can be currently used for bone tissue scaffold fabrication. There are efforts to obtain hybrid scaffolds with proper characteristics by using combinations of natural and synthetic polymers in combination with inorganic materials in the form of hydrogel [
2]. A hydrogel is a three-dimensional unique, soft, and hydrophilic biomaterial [
6], composed of a polymeric network that is able to tightly bind large quantities of water without dissolving [
7]. Due to the high content of water, hydrogels show a flexibility similar to natural tissue. The objective of the presented study was to produce a hydrogel that includes both synthetic and natural as well as inorganic components and thereby to balance the advantages and disadvantages of this scaffold for use in bone tissue engineering. Therefore, the hydrogel scaffold based on polyvinyl alcohol as a synthetic and hyaluronic acid as a natural component, supplemented with hydroxyapatite, was chosen for this study.
Polyvinyl alcohol (PVA) is an often tested synthetic polymer with a good biocompatibility [
8]. It can be transformed into hydrogel easily through the use of repeated freeze-thaw cycles [
9]. This physical crosslinking, based on the reaction of side hydroxyl groups of PVA [
10], is very convenient as there is no need to use any chemical cross-linker that may cause toxicity [
9]. It was found that the number of freeze-thaw cycles, freezing temperature, time, and the concentration of PVA affect structure and the resulting physical [
11] and mechanical properties [
12]. The higher the number of freeze-thaw cycles, the higher the stiffness of the polymer and the loss of PVA chains orientation was observed [
13]. A high hydrophilicity of PVA hydrogels causes suppression of cell adherence to it. However, its intrinsic cell-non-adhesion provides poor support to cell growth and integration to peripheral tissues. PVA could be blended with natural macromolecules, such as chitosan, starch, gelatin, hyaluronic acid, and so on [
10]. Modification of PVA has shown improvements in cell adhesion and growth, for example by hydroxyapatite (HAp). The incorporation of HAp into PVA hydrogel enhanced cell density with good cell spreading morphology.
Hydroxyapatite (HAp), Ca
10(PO
4)
6(OH)
2, as a major natural inorganic component of bone, shows excellent bioactivity, biocompatibility, osteoconductivity, non-toxicity, and non-inflammatory characteristics [
1]. Its mechanical properties are essentially influenced by the size of the HAp particles, porosity, density, etc. [
14]. HAp is very hard but brittle, with a very slow degradation rate in vivo, and that is why it should be joined with natural or synthetic polymers to create scaffolds. On the other hand, HAp is very beneficial for constructing bones, because it promotes the adhesion and proliferation of osteoblasts cultured in vitro [
15]. It stimulates growth factors (e.g., bone morphogenic protein) and elevates activity of alkaline phosphatase (ALP) in mesenchymal stem cells (MSCs) [
1].
Hyaluronic acid (HA) is abundant throughout the ECM, especially connective tissues, and as a structural molecule [
5] in the human body. It is a natural polysaccharide composed of a linear glucosaminoglycan, where repeated units of N-acetyl-D-glucosamine and D-glucuronic acid are linked by alternating β-1,3- and β-1,4- glycosidic bonds [
16]. Not only its biocompatibility and biodegradability but also its viscoelasticity are convenient properties for using HA in biomedicine, health care, and cosmetics. A very significant advantage of HA is its enzymatic degradability by hyaluronidase, an enzyme produced by mammalian cells. In view of the very rapid degradation and water solubility of HA, it is advisable to cross-link it or to blend it with another natural or synthetic polymer [
1].
The aim of our study was to produce a scaffold based on PVA in combination with HA and enriched with HAp and to examine the effect of scaffold composition on the adhesion and proliferation of human osteoblast-like cell line MG-63 cultured in static conditions. According to our knowledge, this combination of all three materials PVA, HA and HAp together was new.