Chapter 1
Structural and Biodegradation Characterization of Supramolecular PCL/HAp Nanocomposites for Application in Tissue Engineering

Parvin Shokrollahi*, Fateme Shokrolahi and Parinaz Hassanzadeh

Department of Biomaterials, Iran Polymer and Petrochemical Institute, Tehran, Iran

*Corresponding author: p.shokrolahi@ippi.ac.ir

Abstract

Conversion of agricultural wastes into biomaterials has been known as a strategy that transforms wastes into high value products. Among different resources of hydroxyapatite (HAp, a major component of bone and teeth), pig bone and teeth, rice bran, cockle shells, and eggshell have received increasingly rising consideration. In this chapter a quick review of agricultural resources of hydroxyapatite is provided. Next, the application of hydroxyapatite in bone tissuce regeneration is discussed. It is shown that in addition to the conventional composite preparation strategies, hydroxyapatite may play a role in advanced materials design and in particular supramolecular nano-composites. Impact of supramolecularly modified hydroxyapatite nanoparticles (SP HAp) on biological activity and mechanical properties of supramolecular polycaprolactone (SP PCL) is discussed. Mechanical measurements on the SP HAp as prepared and the PBS incubated samples verified that formation of PCL clusters around SP HAp slows down the sharp modulus raise and postpones early stage breakage.

Keywords: Supramolecular nano-composite, tissue engineering, renewable resource, hydroxyapatite, polyacprolactone, biodegradation, mechanical properties, biological activity

1.1 Introduction

1.1.1 Hydroxyapatite: A Bioceramic of Renewable Resource

Hydroxyapatite (HAp, Ca10(PO4)6(OH)2); Figure 1.1) is the main inorganic crystalline component of animals' hard tissues including bones and teeth. HAp crystals of bone are generally in the form of needle-like crystals of 5-20 nm in width and 60 nm in length.

Figure 1.1 Crystalline structure of hydroxyapatite.

Hydroxyapatite is well known for hard (bone and teeth) and soft (skin, muscle, and gums) tissues compatibility and has found widespread applications in orthopedic and dental implants as well as hard tissue-engineering scaffolds. Therefore, synthesis and preparation of this bioceramic became the subject of numerous researches either from biological resources (coral, natural bone, etc.), or taking a chemical synthesis approach (Gshalaev & Demirchan 2012). Among the most studied biological resources of hydroxyapatite are pig bone and teeth (generally animals' bone and teeth), rice bran, cockle shells, and eggshell. As an example of HAp preparation from agricultural resources, Fumiaki Yamada et al. have synthesized 200 nm spherical HAp particles from oilless rice bran in two steps. In the first step, the oil less rice bran underwent an acid treatment and then was reacted with calcium salts to yield calcium phytate, which was then calcified, in the second step, at 1000 °C (Yamada 1988). Cockle shells is used as a source of calcium (in the form of calcium carbonate), for hydroxyapatite synthesis, as part of attempts to replace the current synthetic chemistry with clean chemistry (Islam et al. 2013; Lu et al. 2015). In effort to convert the municipal waste stream into materials with added value, eggshell is used as a source of HAp precursor and attracted increasing attention. HAp synthesis strategies from eggshells include heat treatment (Wu et al. 2015, Kamalanathan et al. 2014), hydrothermal (Wu et al. 2013), sol-gel combustion (Choudhary et al. 2015), microwave conversion (Kumar et al. 2012) and precipitation (Goloshchapov et al. 2013). It might be due to this great potential of being used as a source of HAp that eggshell is called "eggshell biomaterial" (Balaz et al. 2015). Also, pigs, bovines, and fish bones as well as pigs' teeth are of main resources of HAp synthesis (Ayatollahi et al. 2015; Mucalo et al. 2015; Piccirillo et al. 2014).

1.2 Biomedical Applications of HAp

Since the concept of tissue engineering was introduced first (Langer et al. 1993), there has been a growing interest in the design of appropriate materials as pure polymers or in the form of blends and composites with bioceramics scaffold materials. Bioceramics are the materials of choice for skeletal repair and reconstruction. Bioceramics, including alumina, zirconia, hydroxyapatite, tricalcium phosphates (TCPs), and bioactive glasses, have made significant contribution to the observed improvement in the quality of human life (Dottore et al. 2014).

Among all of the above-mentioned bioceramics, calcium phosphates and hydroxyapatite, in particular, have been used to repair damaged parts of the musculoskeletal system mainly because of their biocompatibility, low density, chemical stability, and on top of them for their compositional similarity with the mineral phase of natural bone. However, biomedical application of hydroxyapatite is shadowed by insufficient mechanical strength, which results in poor crack growth resistance (in load-bearing applications), and complicated processing; two issues that have been addressed by composite preparation with a number of natural and synthetic polymers in the current literature (Eftekhari et al. 2014; Zhou et al. 2014; Tetteh et al. 2014; Zhang et al. 2014; Baino et al. 2014).

Since 1920, when the first successful medical application of calcium phosphate bioceramics in humans was reported (Albee & Morisson 1920), bioceramics and bioceramic-polymer composites have found numerous applications as replacements of damaged tissues from hips (Ni et al. 2010; Takigami et al. 2010), knees (Shimomura et al. 2014; Matsuo et al. 2015), tendons (Zhao et al. 2014), and ligaments (He et al. 2013; Ge et al. 2012; Shi et al. 2013), to teeth (Wu et al. 2013), periodontal disease, and maxillofacial reconstruction (Pradeep et al. 2012). Recently, efforts have been directed towards improving the biological properties of hydroxyapatite (Ca10(PO4)6(OH)2, HAp) due to outstanding biological responses to the physiological environment. For example, dissolution (Vahabzadeh et al. 2015), bone integration (Yan et al. 2013; Tao et al. 2015), osteogenic differentiation behavior (Li et al. 2015; Elgali et al. 2015), and bioactivity of the HAp particles were studied as a function of chemical composition and in particular when boron or metals such as strontium (Shepherd et al. 2012; Shen et al. 2012), magnesium (Veronesi et al. 2015; Lu et al. 2011), and zinc (Ghorbani et al. 2014) were used as doping agents (Bose et al. 2013).

Effect of HAp particles size on bioactivity was studied by independent research groups, and favorable properties were reported for nano-HAp (nHAp) in biomedical applications (Liu et al. 2013). For example, Cai et al. have prepared nHAps of 20-80 nm in diameter and showed that the greatest cell viability and proliferation of mesenchymal stem cells were observed on 20 nm particles. Meanwhile, the 20 nm particles inhibited the growth of osteosarcoma cells. This group concluded that particle size plays a key role in biological behavior (Cai et al. 2007).

In another study, the effect of nHAp/chitosan seeded with bone marrow mesenchymal stem cells (BMSCs) on bone regeneration was studied in vitro and in vivo, and the results were compared with those observed for mHAp/chitosan composite. It was shown that the scaffolds of nHAp/chitosan induced higher proliferation of BMSCs than mHAp/chitosan. Also, the amounts of the mRNA for BMP-2/4, ALP, collagen I, and integrin subunits increased significantly on nHAp/chitosan as compared to mHAp/chitosan even in osteogenic medium. To investigate the impact of particles morphology on bioactivity, HAp particles of different shapes including rod-like, nano- and micron-sized HAp particles, and ellipse-like nano-HAp particles were prepared and co-cultured with highly malignant melanoma cells using phosphate buffer saline (PBS) as a control. The results indicated that the HAp particle size (nHAp), rather than morphology, performed more effectively in inhibiting the malignant melanoma cells proliferation (Li et al. 2008).

Among effects such as reinforcement of mechanical properties, and bioactivity enhancement, other roles are considered for HAp particles when added in polymer matrices.

For example, it is generally agreed that bioceramics play a role in biodegradation rate of polyesters/calcium phosphate bioceramic composites. The phosphate ions released upon dissolution of HAp are believed to buffer the surrounding medium of biomedical devices and scaffolds (Ehrenfried et al. 2008; Yang et al. 2009). This is especially important in case of devices made of polyesters such as PLLA (poly(L-Lactide), polylactide-co-glycolide (PLGA), and polycaprolactone (PCL) that produce carboxylic acid end groups upon polyester chain cleavage. The buffering effect of HAp slows down the degradation of the matrix polyester through pH neutralization. It has been also reported that the solubility of HAp is increased because of the acidity of the degradation products of biodegradable polyesters and concluded that the calcium and phosphate ions released by the dissolved HAp were helpful in new bone formation (Higashi et al....