Physical and degradation properties of PLGA scaffolds fabricated by salt fusion technique.

Tissue engineering scaffolds require a controlled pore size and interconnected pore structures to support the host tissue growth. In the present study, three dimensional (3D) hybrid scaffolds of poly lactic acid (PLA) and poly glycolic acid (PGA) were fabricated using solvent casting/particulate leaching. In this case, partially fused NaCl particles were used as porogen (200-300µ) to improve the overall porosity (≥90%) and internal texture of scaffolds. Differential scanning calorimeter (DSC) analysis of these porous scaffolds revealed a gradual reduction in glass transition temperature (Tg) (from 48°C to 42.5°C) with increase in hydrophilic PGA content. The potential applications of these scaffolds as implants were further tested for their biocompatibility and biodegradability in four simulated body fluid (SBF) types in vitro. Whereas, simulated body fluid (SBF) Type1 with the optimal amount of HCO3 (-) ions was found to be more appropriate and sensible for testing the bioactivity of scaffolds. Among three combinations of polymer scaffolds, sample B with a ratio of 75:25 of PLA: PGA showed greater stability in body fluids (pH 7.2) with an optimum degradation rate (9% to 12% approx). X-ray diffractogram also confirmed a thin layer of hydroxyapatite deposition over sample B with all SBF types in vitro.

INTRODUCTION

The core idea of tissue engineering is to allow the cells to repair and regenerate damaged tissues and organs by promoting cell growth and differentiation over the scaffolds[1],[2]. These scaffolds are biodegradable matrices designed to support cell proliferation, which finally provides a functional tissue[3]. Since scaffolds are temporary matrices, the degradation performance of the scaffolds must correspond to the regeneration rate of the affected tissues[4]. In this regard, the selection of scaffold material is very important to facilitate the cells to behave in the desired manner to generate tissues or organs of our requirement[5].

The materials used to synthesize biodegradable scaffolds for bone tissue engineering applications ranges between inorganic materials such as ceramics to synthetic polymers. Among them, synthetic polymers have the ability to tailor mechanical properties and degradation kinetics of scaffolds to suit various tissue engineering applications[6]. The intrinsic properties of the polymer materials play a strategic role in the morphology, texture and performances of the scaffold[7]. To be an artificial scaffold, the structure and the surface morphology of the scaffolds have to meet general requirements specific for the targeted tissue: i) three-dimensional architecture; ii) interconnected pores to ensure cell growth, diffusion of nutrients and metabolic waste; iii) suitable surface chemistry; iv) suitable mechanical properties; v) controllable biodegradation and bioresorbability[8],[9].

In the current study, three combinations of hybrid scaffolds were prepared by blending polylactic acid (PLA) and polyglycolic acid (PGA) at the ratios of 80:20, 75:25 and 70:30 and these polymers are known for their cell-based tissue engineering approaches. These polymers have been shown to be degraded mainly by hydrolysis of ester bonds into acidic monomers, which can be removed from the body by physiological metabolic pathways[10],[11] (). The process which we adopted for preparation of microporous biodegradable scaffolds was solvent casting/particle leaching, where we used non-dispersed sodium chloride (NaCl) as particulate porogen for improved pore interconnectivity[12]. In this case, pores were interconnected via fused salt particles prior to the synthesis of three-dimensional (3D) polymer scaffolds. Thus, dissolution of this fused porogen matrix leaves a highly interconnected pore structure in the polymer scaffolds[13].

Fig. 1

Structure of PLGA molecule.

The figure shows hydrolytic degradation to PLA and PGA monomers in the presence of physiological fluids. PLGA: poly (lactic co glycolic acid).

Once the polymer scaffolds are made, there is an immediate need to test the scaffolds for both in vivo and in vitro in order to consider them for human applications[14],[15]. These studies include their physical, chemical and mechanical properties helpful for assessing their bioavailability[16]. In case of in vitro studies, scaffolds were exposed to a group of model solutions simulating the inorganic portions of blood plasma to study their surface interaction and changes. The composition of the most used simulated body fluids differs from that of human blood plasma by high content of Cl- and lower content of HCO3− ions. Considering the composition of bone like apatite, which contains carbonate ions, the test results could be influenced by this difference[17],[18]. In this study, we prepared four different simulated body fluids with varied concentrations of the above said ions and we monitored the influence of these simulated fluids on the physico-mechanical properties of polymer scaffolds.

MATERIALS AND METHODS

Scaffold preparation

PLA (2.9 kg/mol) and poly glycolic acid (PGA) (IV= 1.2 dL/g) were procured from Sigma (St. Louis, MO, USA). Porous polymer scaffolds were prepared by solvent casting/particulate leaching where we used NaCl salt as particulate porogen (200-300 µm). PLA and PGA with higher molecular weight/inherent viscosity were used in our studies to ensure that the scaffolds would hold adequate mechanical integrity despite their relatively high porosity (≥90%). Briefly, NaCl matrices were prepared by subjecting NaCl particles to 95% humidity for 12 hours prior to solvent casting. PLA and PGA were blended at the ratios 70:30, 75:25 and 80:20 and dissolved in Hexafluoro-2-propanol (Sigma). This molten polymer blends were poured in to non-dispersed NaCl matrices (or) scaffold before solvent evaporation. Then, these scaffolds were vacuum dried for 48 hours before NaCl particles were further leached out by immersing scaffolds in de-ionized water.

Scaffold characterization

Electron microscopy

The transverse sections of NaCl scaffolds were imaged using scanning electron microscope (SEM) prior to solvent casting to monitor NaCl crystal fusion. In addition, transverse sections of polymer scaffolds after salt leaching were also imaged using SEM (Zeiss EVO® MA15).

Determination of glass transition temperature (Tg)

Glass transition temperature (Tg) of all three scaffold types was determined as per ASTM D7426 standard by differential scanning calorimeter (DSC) equipped with liquid nitrogen cooling system (Auto Q20, TA instruments). Ten mg of polymer samples were quantitatively transferred to sealed aluminum pans and subjected to cooling and heating cycles from 0°C to +200°C with cooling and heating rates of 5°C/min. During experiment, DSC cell was purged with dry nitrogen at 40 mL/min. The baseline correction was performed by recording a run with empty pans.

Preparations of simulated body fluids

We tested the polymer degradation as per ASTM F1635-04a standard with four simulated body fluid types by varying Cl− and HCO3−[19]. The composition of simulated body fluids is shown in . SBFs were prepared in polypropylene beakers by dissolving NaCl, NaHCO3, KCl, K2HPO4, MgCl2, 1M HCl, CaCl2, Na2SO4, Tris HCl in double distilled water and pH was adjusted to 7.4 and the fluids were further incubated at 37°C for 3 to 4 days and monitored for hydroxyapatite (HA) deposition.

Table 1

The composition of inorganic components required for the preparation of simulated body fluids (mmol/L)

	SBF1	SBF2	SBF3	SBF4
Na+	142.0	142.0	142.0	142.0
K+	2.0	2.0	2.0	2.0
Ca2+	2.5	2.5	2.5	2.5
Mg2+	1.0	1.0	1.0	1.0
Cl−	116	121.0	126.0	131.0
HCO3−	20.0	15.0	10.0	5.0
SO42−	1.0	1.0	1.0	1.0
HPO42−	1.0	1.0	1.0	1.0

In vitro degradation of scaffolds by immersion method (ASTM F1635-04a)

Polymer scaffolds were cut to uniform sizes and each sample was weighed before the immersion test[20]. Then, scaffolds were placed in separate polypropylene beakers and fully immersed in simulated body fluids (0.2 mL of SBF/mm3 of scaffold) and incubated for 21 days at 37°C. These polymer scaffolds were taken out at preferred time intervals (at 14 and 21 days) and rinsed with distilled water and dried further for studying the morphological changes and weight loss; simultaneously, we checked for pH shift in simulated body fluids due to polymer degradation.

Analysis of the scaffold surface by X-ray diffraction

The interaction of polymer scaffolds with body fluids was evaluated by studying surface modifications over the scaffolds by X-ray diffraction (XRD) analysis. Precisely, polymer scaffolds immersed in four different simulated body fluids for 21 days were further vacuum dried in order to test HA deposition over the scaffold surface using XRD (Shimadzu) with a 2 theta (2θ) angle between 10 to 80 degrees at a scan speed of 5°/min.

RESULTS

Highly porous (≥90%) hybrid poly (lactic co glycolic acid) (PLGA, scaffolds (3 mm thickness) were prepared by the solvent casting/particulate leaching method. These scaffolds were cut to uniform sizes for further characterization. For our convenience, scaffolds with 80:20, 75:25 and 70:30 of PLA: PGA are denoted as Sample A, Sample B and Sample C respectively.

Initial incubation of NaCl crystals in a humidifier (95%) resulted in fusion of salt crystals, creating interconnected matrices (). This fused NaCl crystals prior to addition of molten polymer mixture increased pore interconnectivity, which improved the overall porosity of scaffolds (≥90%) ().

Fig. 2

Scanning electronic microscopic images of fused salt crystals (A) and porous polymer scaffolds (B).

The DSC analysis of PLGA scaffolds revealed their amorphous nature identified by the presence of glass transition temperature (Tg) and by the absence of melting temperature (Tm). The PLGA scaffolds showed increased polymer degradation with increases in the PGA proportion in the scaffolds. We also noticed a gradual reduction in Tg of polymer scaffolds A, B and C to 48°C, 44.5°C and 42.5°C, respectively.

In vitro degradation of scaffolds by the immersion method (ASTM F1635-04a)

1) Morphological variations with time

After 14 days in simulated body fluids, the morphological changes were found to be irregular, with increase in pore size over the scaffold surface. After 21 days, regular and recognizable morphological changes were detected in PLGA scaffolds. We also compared the morphological variations with weight loss in PLGA scaffolds during degradation studies.

2) Weight loss

Sample C had shown accelerated weight loss when compared to the other two samples; this may be due to higher PGA content in the scaffold, which is hydrophilic in nature. Though the percentage degradation was high in case of samples C, the degradation rate aws not even (approximately 11% to 22%). This uneven degradation property makes this combination inappropriate for in vivo application. Comparatively, sample B had a stable and optimum degradation rate (approximately 9% to 12%), whereas sample A exhibited a low degradation rate (approximately 4% to 8%) compared to the other two samples. These observations indicated that increase in the PLA content made the scaffolds more hydrophobic and denser, thus making them tougher to be degraded both in vivo and in vitro.

3) pH shift in the simulated body fluids over time

By the end of 30 days of incubation, shows the pH shift in the simulated body fluids over time at 37°C. Sample A showed a slight increase in pH from 7.4 to 7.6, which might be due to slower polymer degradation and continues release of (PO4)3- ions which acts as conjugate bases. Sample C, due to its accelerated degradation to lactic and glycolic acids; showed higher decline in pH from 7.4 to 6.7, making this sample vulnerable for in vivo applications whereas sample B was found to be stable with a shift in pH from 7.4 to 7.2.

Fig. 3

Time dependence on pH of simulated body fluids.

A: 80:20 PLGA; B:75:25 PLGA; C: 70:30 PLGA. And 2, 3 and 4 represents four SBF types

4) Analysis of scaffold surface by XRD

XRD analysis using the X-ray diffractometer revealed the presence of straight base line and semi-sharp peaks (), suggesting the semi crystalline nature of our PLGA scaffolds. The XRD patterns also clearly indicated the deposition of HA traces over the scaffold surface. Compared to other SBF types, the XRD spectrum of SBF1 showed better HA deposition whose carbonate content and phosphate contents are more similar to human blood plasma compared to other SBF solutions.

Fig. 4

XRD analysis of polymer scaffolds for HA deposition.

A: XRD spectrum of Pure HA. B: XRD spectrum of Sample A. C: XRD spectrum of Sample B. D: XRD spectrum of Sample C.

DISCUSSION

Porous hybrid polymer scaffolds (PLGA) were prepared by the solvent casting/particle leaching method, where we fused NaCl particles by prolonged exposure to rich moist environment (95% humidity), resuling in enhanced pore interconnectivity in the PLGA scaffolds (). In this study, fused NaCl particles resulted in the creation of holes on the walls of the scaffolds, which increased the comprehensive modulus of the polymer scaffolds[21],[22]. Improved pore interconnectivity is also helpful in a variety of tissue engineering applications, particularly those requiring close cell to cell contact[23].

Fig. 5

Micro structure of highly porous PLGA scaffolds.

Whereas A depicts fused NaCl particles with contact points resulted in salt bridges between the particles; B: SEM images of the polymer scaffolds exhibit optimum porosity (≥90%).

depicts the transverse section of PLGA scaffolds prepared using fused salt particles as porogen. Scanning electron micrographs illustrate the polymer scaffolds with highly porous and well interconnected network. The microstructures of the scaffold determine its interaction with the cells and molecular transport of nutrients and biological wastes from within the scaffold[24]. Exclusively, the pore size of the scaffolds determines the cell seeding efficiency into the scaffold; small pores prevent the cells from piercing into the scaffold, while very large pores prevent cell adhesion due to reduced area to colonize cells[25],[26]. Therefore, scaffold with an open and interconnected pore network and high degree of porosity (≈90%) is described as a perfect model to integrate with the host tissue[27].

PLGA scaffolds prepared by salt fusion also showed irregular pore sizes, ranging between few microns to 300 µm. This variation in porosity may be due to a phenomenon known as solid-liquid phase separation which is attributed to solvent crystallization[28]. When the temperature of the polymer solution is lower than the solvent freezing point (crystallization temperature), solvent crystallizes and the polymer phase is expelled as impurity. A continuous polymer-rich phase is formed by the aggregation of polymer fractions excluded from solvent crystals[29]. After solvent crystals have been sublimated, the scaffold is produced with a micro-porosity similar to the geometry of solvent crystals.

In in vitro degradation studies, PLGA scaffolds were degraded by hydrolysis of their ester linkages[30]. The presence of methyl side chain in PLA makes it more hydrophobic and denser than PGA and hence lactide-rich PLGA copolymers are less hydrophilic, absorb less water and are subsequently degraded at a lower rate[31],[32]. Additionally, reduced molecular weight with increased PGA content influences the reduction in “Tg” of the PLGA scaffolds (from 48°C to 42.5°C, which was quite near the incubation temperature of 37°C)[33],[34]. All the above discussed features made sample B (PLA: PGA (75:25)) as a favorite in comparison with other scaffolds. Also, controlled degradation (9-12%) of sample B might provide the room for tissue growth both in vivo and in vitro as biodegradable or restorable material.

X-ray diffraction spectra of polymer scaffolds after interaction with different model solutions are explained in . After 21 days of immersion in simulated boy fluids, a thin layer of HA deposition was observed over the scaffold surface. Comparatively, SBF1 showed characteristic peaks for HA with all three scaffold types. This fact indicates that SBF1 with carbonate content similar to the human blood plasma could be more suitable and sensitive for in vitro testing of bioactivity and the diffusive character of observed peaks might be the result of poor crystallinity of the precipitated product due to the relative short time of exposure in the simulated body fluids and/or thinner precipitated layer[35]. The slower apatite deposition with SBF1 in comparison with other body fluids could enable the more sensitive in vitro testing of bioactive materials. Moreover, during interaction with human blood plasma, the creation of carbonated hydroxyapatite could be awaited rather than pure HA precipitation. Therefore, the content of carbonate ions in the solutions can be important for the plausibility of in vitro test.

In conclusion, in vitro degradation behaviors of PLGA scaffolds in three different formulations were tested systematically with four simulated body fluids for 30 days. Detailed quantitative studies on the physiological features of scaffolds in wet environment along with other material parameters were tested. During these studies, sample B was found to be more appropriate with better physiological characteristics for further in vivo studies. The composition of SBF1 also proved as a better source for further optimization studies.