Preparation and Characterization of PLLA CompositeScaffolds by ScCO2-Induced Phase Separation

Zhaohong Ding,1,2 Zhijun Liu,1 Wei Wei,1 Zhiyi Li11Institute of Fluid and Power Engineering, School of Chemical Engineering,Dalian University of Technology, Dalian 116024, China

2Department of Shipboard Weaponry, Dalian Naval Academy, Dalian 116008, China

Composite Scaffolds have received much attention inthe tissue engineering, and how to choose the materialshas become the research focus in this field. Supercriti-cal CO2 (ScCO2)-induced phase separation process wasemployed to prepare porous poly-L-lactide (PLLA) com-posite scaffolds. An experiment system was set up forthe purpose of investigating the effects of such parame-ters as the mass ratios of PLLA to polyethylene glycol(PEG) and PLLA to b-TCP on porosity and compressivestrength of composite scaffolds. The obtained compos-ite scaffolds were characterized in many ways. Scan-ning electron microscopy was used to examine themorphology and pore size; porosity was analyzed bypycnometer; and the compressive strength wasrecorded by texture analyzer. The results indicated thatthe porosity was increased with the addition of PEG,and the highest porosity of PLLA/PEG composite scaf-folds was 92% with the mass ratio of PLLA to PEG of1:0.05; the compressive strength was increased with theaddition of b-TCP, and the highest compressive strengthof PLLA/b-TCP composite scaffolds was 1.76 MPa withthe mass ratio of PLLA to b-TCP of 1:0.1. POLYM. COM-POS., 00:000–000, 2012. ª 2012 Society of Plastics Engineers

INTRODUCTION

The selection of scaffold materials has been the

research focus due to its great potential in tissue engineer-

ing systems. Many natural and synthetic polymers have

been proposed for scaffolding applications. Poly-L-lactide

(PLLA) is biodegradable aliphatic polyester, which is

derived from renewable resources and is considered to be

one of the most promising polymers as scaffold materials.

However, a single PLLA porous scaffold material has

some shortcomings such as less hydrophilicity, acidic

degradation products, and the poor compressive strength.

Combining PLLA with other polymers to form composite

scaffolds is one way to solve the problems [1–3]. Com-

posite scaffolds, which are made of two or more comple-

mentary materials can overcome the shortcomings of a

single material and can maintain the advantages of pure

materials. It has gained more and more interests in recent

years [4–6].

Many methods have been developed to prepare com-

posite scaffolds, including porogen leaching [7], thermally

induced phase separation [8], freeze-extraction, freeze-

gelation methods [9], and so on. Although these methods

have some advantages, they still have some limits that

cannot be overcome, such as organic solvent residue, low

porosity, and poor compressive strength.

Supercritical CO2 (ScCO2)-induced phase separation

process has been recently proposed to prepare porous

materials. Compared with other methods, it has some

unique advantages: ScCO2 can dry the porous structure

rapidly without causing collapse due to the absence of a

liquid–liquid interface; the process does not require addi-

tional post-treatments and the solvent dissolved in ScCO2

can be easily removed and recovered. Although some

researches have been conducted in porous structure for-

mation by ScCO2-induced phase separation process [10–

13], the materials used in these studies are single pure

materials, such as polystyrene, polycarbonate, nylon6, and

so on.

Polyethylene glycol (PEG) has been widely used as a

biomedical material [14, 15] for its good blood compati-

bility, hydrophilicity, water solubility, and non-immuno-

genicity. b-Calcium phosphate (b-TCP) can be absorbed

and replaced by new bone [16]. Preparations of PLLA/

PEG and PLLA/b-TCP scaffolds have been reported [17,

18], but neither used ScCO2-induced phase separation

process.

In our previous work, the micro-porous poly-vinylbuty-

ral membranes were successfully prepared by ScCO2-

induced phase separation process [19]. The main purpose

Correspondence to: Zhiyi Li; e-mail: lizy@dlut.edu.cn

Contract grant sponsor: National Nature Science Foundation of China;

contract grant number: NSFC 30870646.

DOI 10.1002/pc.22299

Published online in Wiley Online Library (wileyonlinelibrary.com).

VVC 2012 Society of Plastics Engineers

POLYMER COMPOSITES—-2012

of this work is to investigate the feasibility of preparing

PLLA/PEG and PLLA/b-TCP composite scaffolds by

ScCO2-induced phase separation process. The porous

structures of pure and composite PLLA scaffolds are pre-

pared under the same operation procedures. The effect of

PEG and b-TCP content on porous structures is investi-

gated, and the porosity and compressive strength of the

composite scaffolds are examined as well.

MATERIALS AND METHODS

Materials

PLLA (molecular weight 150,000) was obtained from

Shandong Dai Gang Biological Technology. b-TCP

(diameter 20 nm) was provided by Beijing Hua Ken En-

sign Technology. PEG (molecular weight 3,600–4,000)

and dichloromethane (purity>99.5%) were purchased from

Tianjin Bodi Chemical. CO2 (purity, 99%) was kindly

provided by School of Chemical Engineering, Dalian

University of Technology. All materials were used as

received.

Composite Scaffolds Preparation

The experimental setup is schematically shown in

Fig. 1. PLLA powders with different mass ratios of

PEG and b-TCP were dissolved in dichloromethane and

the solution was homogenized by a magnetic stirring.

After removing air bubbles, the solution was injected in

a solution container (stainless steel cap with 4.5 cm

diameter) installed in the high pressure cell (internal

volume 500 mL), which was placed in an isothermal

water bath. The high pressure cell was sealed and com-

pressed CO2 was introduced into the cell by an air com-

pressor until the desired pressure was reached. After

this, CO2 cylinder, valve 2, and compressor were closed

in turn. The system was held for 3 hr at the constant

pressure and temperature to get ScCO2 and dichlorome-

thane dissolved mutually and thoroughly. Then valves

2 and 3 were opened to sweep the cell with fresh com-

pressed CO2 at the same conditions to extract the resid-

ual organic solvent. At the end of the experiment, the

cell was slowly depressurized with the experimental

temperature unchanged. The depressurization time was

over 1 hr.

FIG. 1. Schematic presentation of the experimental setup used for the

preparation of composite scaffolds by ScCO2-induced phase separation

process.

FIG. 2. SEM morphology of PLLA/PEG scaffolds showing the porous structure at different mass

ratios: (a) 1:0; (b) 1:0.04; (c) 1:0.05; and (d) 1:0.065.

2 POLYMER COMPOSITES—-2012 DOI 10.1002/pc

Properties and Characterization

The porous scaffolds were freeze-fractured in liquid

nitrogen and sputter coated with gold, their cross-sections

were examined by scanning electron microscopy (SEM,

KYKY-2800B). The porosity of scaffolds was measured by

pycnometer method [20]. The compressive strength of scaf-

folds was determined by texture analyzer (TMS-PRO).

RESULTS AND DISCUSSION

To examine the feasibility to prepare PLLA composite

scaffolds by ScCO2-induced phase separation process, the

experiments were performed with two different additives,

PEG and b-TCP.

Composite Scaffolds of PLLA/PEG

A series of experiments were conducted under the con-

dition of PLLA concentration of 6% (wt/wt), CO2 pres-

sure of 10 MPa, temperature of 508C and different mass

ratios of PLLA to PEG ranging from 1:0 to 1:0.065. The

effect of mass ratio on the scaffolds structure of the

cross-section is shown in Fig. 2. It could be seen that the

mean pore size (76.4 lm, 79.6 lm, 82.2 lm, and 89.2

lm) increased gradually with the increase of PEG con-

tent. Figure 3 shows the diameter distributions of pores

on cross-sections obtained from different mass ratios,

which confirmed the qualitative observations obtained

from Fig. 2. A great difference between the pure porous

material and composite one is shown in Fig. 2a and Fig.

2b–d, respectively. The pure polymer (Fig. 2a) exhibited

more regular structure than that with the addition of PEG

(Fig. 2b–d). It was shown that the process induced by

ScCO2 phase separation was helpful in forming irregular

FIG. 3. Cross-section pore size distribution of the PLLA/PEG scaffolds

at different mass ratios: (a) 1:0; (b) 1:0.04; (c) 1:0.05; and (d) 1:0.065.

TABLE 1. Character of PLLA/PEG scaffolds.

mPLLA:mPEG

Mean

porosity (%) 6 SD

(n ¼ 3)

Mean compressive

strength (MPa) 6 SD

(n ¼ 3)

1:0 80 6 0.56 1.17 6 0.03

1:0.04 84 6 0.76 1.20 6 0.02

1:0.05 92 6 0.89 1.22 6 0.04

1:0.065 81 6 0.51 1.12 6 0.03

FIG. 4. SEM morphology of PLLA b-TCP scaffolds showing the porous structure at different mass

ratios: (a) 1:0.05; (b) 1:0.06; (c) 1:0.1; and (d) 1:0.2.

DOI 10.1002/pc POLYMER COMPOSITES—-2012 3

cellular structure with rough surface and micro-grooves,

which were more conducive to cell adhesion, orientation,

migration, expansion, and proliferation [21].

The effect of the PEG mass ratio on porosity and com-

pressive strength is shown in Table 1. With the increase

of PEG mass ratio, the porosity of composite scaffolds

increased from 80% without PEG to 84% with PEG mass

ratio of 1:0.04, and reached the highest of 92% with the

mass ratio of 1:0.05, and the compressive strength had no

obvious change. After the phase separation, fresh CO2

injected in the cell could not only take away CH2Cl2, but

also dissolve PEG, which had increased the pore connec-

tivity and porosity of the scaffold. Even if any residual

PEG remained in the scaffolds, it may not affect biomate-

rial applications due to its biocompatibility. With the

further increase of PEG mass ratio, cross-section of the

scaffold became more irregular (Fig. 2d) and blocked

the connectivity among holes to a certain extent, so that

the porosity of the scaffolds decreased.

Composite Scaffolds of PLLA/b-TCP

Composite scaffolds were prepared with the mass ratio

of PLLA to b-TCP from1:0.05 to 1:0.2 (the initial PLLA

concentration was constant of 6 wt%) at 508C and 10

MPa. Figure 4 shows the cross-sectional structure of com-

posite scaffolds of PLLA/b-TCP measured by SEM. It

could be seen that the b-TCP particles dispersed homoge-

neously in the scaffolds. The uniform b-TCP in the scaf-

folds can enhance the strength of scaffolds from three-

dimensional direction. The diameter distributions of pores

on cross-sections obtained under different mass ratios are

shown in Fig. 5. With the increase of the mass ratio from

1:0.05 to 1:0.1, the mean diameter increased from 84.5 to

107.4 lm. With the further increase of b-TCP mass ratio,

the mean diameter of the scaffold decreased to 80.2 lm

(Fig. 4d). On the other hand, with the increase of the mass

ratio of PLLA to b-TCP, the porosity changed a little

(Table 2). The reasons may be that the increase of b-TCP

had no effect on the formation of the PLLA continuous

phase.

At the same time, Table 2 showed that the compressive

strength of PLLA/b-TCP scaffold was changed with the

increase of the b-TCP mass ratios. The compressive strength

of pure PLLA scaffold was 1.17 MPa. When the mass ratio

of PLLA to b-TCP was 1:0.1, the pore size became very

uniform and the compressive strength increased to 1.76 MPa.

When the mass ratio of PLLA and b-TCP was 1:0.2, the

compressive strength decreased to 1.23 MPa. The reason for

this might be that the excess of b-TCP in the PLLA scaf-

folds resulted in more irregular pores, which caused a more

severe stress concentration [22].

CONCLUSIONS

PLLA composite scaffolds were successfully prepared

by ScCO2-induced phase separation process. In the pre-

pared composite scaffolds of PLLA/PEG and PLLA/b-

TCP, the PEG and b-TCP could disperse homogeneously

in PLLA continuous phase matrix. By changing the PEG

and b-TCP mass ratio, the porosity and compressive

strength of composite scaffolds could be adjusted. With

the increase of the PEG mass ratio, the porosity increased.

The highest porosity of PLLA/PEG composite scaffolds

was obtained at the mass ratio of PLLA to PEG of

1:0.05. With the increase of the b-TCP, the compressive

strength increased and the PLLA/b-TCP composite scaf-

folds had the highest compressive strength when the mass

ratio of PLLA to b-TCP was 1:0.1.

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