Studies on enzymatic degradation of multifunctional composite consisting of chitosan microspheres and shape memory polyurethane matrix

Highlights

• In vitro degradation studies of 3b-PU/CH-M composite consisting of chitosan microspheres (CH-M) embedded in polyester-urethane matrix were performed in PBS/Lysozyme medium.

• Three stages of the degradation of 3b-PU/CH-M composite were distinguished over 24 weeks.

• Morphology changes of CH-M in the outer layer of the composite film and no signs of degradation of CH-M in deeper layers of the composite was observed.

• Adhesion of lysozyme on the composite surface and inhibition of mass loss (%) of the composite was observed after 8 weeks of degradation.

Abstract

Degradation studies of the multifunctional 3b-PU/CH-M composite consisting of 2.5 (wt.%) chitosan microspheres (CH-M) incorporated into crosslinked poly(caprolactone/lactide-co-glycolide) (PCL/PLGA) - urethane matrix (3b-PU) were performed in vitro in phosphate buffered saline (PBS) with lysozyme (Lys). The degradation process of the composite was monitored and analyzed via scanning electron microscopy (SEM), weight loss, pH measurement, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA-FTIR). FTIR analysis of chemical structure and mechanical properties measurement were also performed. Three stages of degradation were distinguished during 6 months studies: first - lasting the first 4 weeks associated with morphology changes of CH-M embedded in the outer layer of the composite film and 3% mass loss, followed by significant mass loss up to 20% and pH decrease to 6.5 attributed to degradation of PLGA units observed between 4 and 8 weeks of degradation. The last stage that was characterized by lysozyme adhesion on the composite surface and mass loss inhibition. SEM observations revealed that CH-M dispersed in the deeper layer of the polymer matrix preserved 6 months of degradation in PBS/Lys solution. In vitro test on MG-63 cells revealed no significant effect of the degradation products on cell viability and proliferation.

Introduction

Today's advanced biomedicine is driven by innovations in material science [1]. Among many different groups of materials, polymers are predominantly utilized in medical devices and pharmaceutical applications [1], [2], [3]. Biodegradable implantable polymers play a significant role especially in regenerative medicine [4]. The modern biopolymers are increasingly expected to perform a multiple functions like mechanically support newly formed tissue, reshape after implantation and therefore enable to use minimally invasive surgery procedures, degrade gradually within a certain period of time, break dawn into nontoxic products, as well as deliver therapeutic agents [5,6].

Degradability in physiological environment [7] is one of the most important function of multifunctional materials for biomedical purpose. Fabrication of resorbable implants for tissue engineering requires to use advanced materials which undergo in vivo degradation and bioresorption in a controlled manner over a predetermined time. This approach allows to avoid chronic immune response to the presence of the implant and additional surgery to remove the implant [7], [8], [9]. Biopolymer constructs can also deliver specific drugs with different therapeutic effects, such as antibacterial, anti-inflammatory, stimulating cellular proliferation and differentiation [10], [11], [12], [13]. Since most of the drugs exploited in tissue engineering are characterized by pleiotropic nature, it is crucial to release them in a sustainable way [14,15]. For example typical drug-loaded implants from biodegradable polymers are systems consisting of drug dispersed within biodegradable polymer matrix and exhibit two functionalities: the capability of drug molecules to gradually escape from the matrix for a controlled release and the ability of the matrix to subsequently degrade for complete excretion from the body. In such systems, drug release can be both diffusion and erosion controlled as the polymer matrix acts as a diffusion barrier to slow the release of drug, release rates can increase as that barrier degrades [16,17]. Therefore, the knowledge of the degradation kinetics is crucial when designing new biomaterials with specific biodegradable characteristic for their safe use in biocomponents [18,19].

Among various polymeric biomaterials, biodegradable shape memory polyurethanes (PU) have been developed for a wide range of biomedical applications due to their versatility and biocompatibility [20], [21], [22], [23], [24], [25], [26]. Biodegradable PU usually possess a segmented polymer network composed of soft segments comprising polyether or polyester crosslinked with urethane bonds and hard segments formed between isocyanate and chain extender [21]. Variations in the degradation patterns of various kinds of PUs results from a differences in polymer network architecture such as molecular orientation, crystallinity, cross-linking, and chemical groups presented in the molecular chains which determine the accessibility to water and enzymes [22,23]. In case of polyether based soft segments the underlying mechanism of degradation in vivo was considered to involve oxidative degradation [24], whereas PU based on polyester soft segments such as polycaprolactone (PCL), polylactide (PLA), polylactide-co-glycolide (PLGA) or its copolymers and mixtures undergo hydrolysis, in which enzymes may also play an important role (i.e. enzymatic degradation) [25], [26], [27]. Polycaprolactone based PU are characterized by the slowest degradation among polyester-based PU due to semi-crystalline nature of PCL which hindrance water diffusion and thus decrease the hydrolytic degradation rate and disturb the drug release profile. On the other hand, PCL crystallinity is the feature essential for thermoresponsive shape memory polyurethanes (SMPU) microstructure. Shape memory capability, an additional function of the implantable devices, is a phenomenon of restoring the original memorized shape of a plastically deformed sample upon exposure to an external stimulus [28], [29], [30], [31]. PCL semi-crystalline switching segments may undergo thermally reversible changes of softening and hardening above melting temperature and therefore provide temporary shape fixation [32]. Therefore, when designing shape memory biodegradable polyurethanes based on PCL semi-crystalline segments the polymer network architecture should be carefully balanced to tune degradation rate, shape memory capacity and drug delivery. Our previously reported study on in vitro biodegradation of multifunctional, shape memory electrospun PU comprising PCL switching segments preformed in PBS revealed that the incorporation of low molecular weight, hydrophilic PEG segment or amorphous PLGA units to polymer backbone increase degradation rate and drug release. Additionally, more predictable changes of the mechanical properties and better shape memory performance exhibited PU based on the mixture of PCL and PLGA than obtained from PCL/PEG/ [33].

Recently, we have developed a multifunctional composite material, based on crosslinked PCL/PLGA-urethane matrix with shape memory properties and chitosan microspheres that provide the final material system with drug delivery capability as additional function. Chitosan microspheres were used as drug carrier due to its outstanding biocompatibility, biodegradability, and antibacterial properties [34], [35], [36]. It was reported that chitosan degradation occurs mainly in the presence of lysozyme, which commonly exists in various human body fluids and tissues [27,[37], [38], [39]]. Chitosan degradation undergoes through breakdown of the glycosidic bonds [40]. It was also recognized that degradation rate of chitosan is influenced by many factors such as deacetylation degree, crosslinking density, crosslinking agent, or molecular weight [41].

Although, the biodegradation ability of PU, polyesters and chitosan microspheres alone have been well characterized and widely reported in the literature [42,43], there is a lack of comprehensive study on degradation of PU/chitosan composite materials especially in the presence of Lys [44,45].

In the present study we have focused on the analysis of biodegradation profile of the multifunctional polyester-urethane/CH-M composite, considered as an additional function of the composite. With respect to the factors that activate degradation of chitosan microspheres and polyester-urethane matrix the experiments were conducted in PBS buffer solution in the presence of lysozyme. The physicochemical and thermomechanical characteristic, as well as shape memory properties, drug delivery capability and biocompatibility of the newly developed biomaterial were thoroughly studied and describe elsewhere [46]. The biodegradation was monitored by following weight loss, changes in pH, chemical structure, as well as microstructural observation. Additionally, to evaluate potential toxicity effect of degradation products in vitro cytotoxicity test with MG-63 cell line was performed.