Jin Zhanga,
Shu-Gui Yanga,
Jian-Xun Dingb and
Zhong-Ming Li*a
aCollege of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, Sichuan, P. R. China. E-mail: zmli@scu.edu.cn
bKey Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China
First published on 9th May 2016
A significant challenge in bone tissue engineering is the development of biomimetic scaffolds that can meet the requirements of mechanical and degradation properties at the same time. The composite scaffolds comprising poly(L-lactide) (PLLA), poly(lactide-co-glycolide) (PLGA) and hydroxyapatite (HA) were fabricated by a new method, i.e., high-pressure compression molding plus salt-leaching technique. The scaffolds obtained show an encouraging improvement in the mechanical performance. The compressive modulus reaches up to 4.64 ± 0.2 MPa, comparable to human cancellous bone (2–10 MPa), offering the possibility to develop load-bearing scaffolds. Furthermore, by adjusting the weight ratio of PLLA to PLGA, the degradation rate, hydrophilicity, and mechanical properties of scaffolds can be fine-tuned. The overall characteristics of porous composite scaffolds are definitely optimal when the mass ratio of PLLA/PLGA is 5:
5. Its porosity, contact angle, compressive modulus and weight loss at the 12th week are 81.7%, 53.13°, 4.64 ± 0.2 MPa and 67.21 ± 3.14%, respectively, satisfying the physiological demands to guide tissue regeneration. Scaffolds with the best comprehensive properties are further utilized for in vitro tests. As presented from the excellent spreading and the high proliferation rate of cells, the design of such tailor-made scaffolds as a function of composition is a convenient strategy to address the specific requirements of the tissue to be regenerated.
As a general rule, the degradation rate of scaffolds is mainly determined by the structure, crystallinity and molecular weight of the scaffold material.3 Poly(L-lactide) (PLLA) is the most attractive candidate for scaffold material. It is approved by the Food and Drug Administration (FDA) and can be easily processed into various structures of 3D matrices.4 As shown by the results of in vitro experiments, PLLA scaffold had the highest survival rate of cells compared with other polymers, which was mainly contributed to its high compressive strength and modulus.5 Nevertheless, the chemical structure of PLLA contains an ester bond that is less likely to undergo hydrolysis, so it is a little hydrophobic and degrades slowly (typically 30–50 weeks).6 Poly(lactide-co-glycolide) (PLGA), a new synthetic polypeptide formed by polyglycolic acid and polylactic acid, showing good hydrophilicity, fast degradation rate and being absent of immunogenicity. As a prospective construct material for tissue engineering, the only drawback of PLGA is too flexible, which seriously limits its applications for bone repair materials.7 Given that PLLA and PLGA make a pair of synthetic polypeptide with complementary features, combination of advantages of the two biomaterials will realize some desired properties prior to one of them alone.8 More importantly, the degradation rate of scaffolds could be fine-tuned by adjusting the ratios of PLLA to PLGA. Langer et al. once examined the use of PLLA/PLGA scaffolds for promoting hES cell growth and differentiation and formation of a 3D vessel-like network structure. They claimed that the PLLA/PLGA scaffolds not only possessed a suitable biodegradability, but also could be designed to resist contraction under the compressive stress exerted by the cells.9 Nevertheless, to the best of our knowledge, few investigations once reported systematically on the comprehensive performance of PLLA/PLGA blend for tissue-engineering scaffolds as of yet. Furthermore, fabrication of such scaffolds with tailored compositions, mechanical properties, as well as a proper degradation profile, is essential to mimic the native extracellular matrix and to positively affect the cell behaviour.
Although PLLA or PLGA has been widely accepted as tissue engineering scaffold, they also face the problem of no natural cell recognition sites. As the major inorganic component of bone, hydroxyapatite (HA) has proved capability to promote differentiation of stem cells towards osteoblastic lineage.10 Recently, Gibbons et al. presented a systematic characterisation of bone tissue scaffolds fabricated via 3D printing from HA and poly(vinyl)alcohol (PVOH) composite powders.11 Zhang et al. developed the porous chitosan-HA scaffolds as a mimic of glioblastoma microenvironment ECM.12 In a word, incorporation of HA into PLLA/PLGA blend lays a particular foundation on improving the scaffold bioactivity and osteoconductivity. Additionally, in order to maintain the integrity of the porous structure during the tissue regeneration, scaffolds with enough mechanical strength are actively pursued. Current techniques for attaining porous polyester/HA scaffolds mainly include salt leaching, phase separation, gas foaming, emulsion freeze-drying, and rapid prototype, to name just a few. Nevertheless, the scaffolds fabricated by above-mentioned methods are difficult to engineer clinically useful tissues and organs, which is attributed to the fatal drawbacks of inadequate mechanical properties, especially in the high load sharing situations.13 Compared with these existing methods, the combination of high-pressure compression molding plus salt leaching technique is able to acquire the scaffold with a more compact interpenetrating network structure, an optimized crystalline architecture and an enhanced mechanical property. Based on our previous research work, the storage modulus of PLA/HA scaffolds (87.6 MPa) achieved almost three times higher compared with pure PLA scaffolds, while under low-pressure condition, the increase of modulus caused by HA does not reach 150%. The obvious contrast indicated that HA and high-pressure had a synergistic effect on enhancing mechanical properties of porous scaffolds.14 On this context, such effective method was continued to be utilized for the preparation of PLLA/PLGA/HA composite scaffolds. In the current study, we first discussed the effect of PLLA/PLGA weight ratios on scaffold properties in detail, with respect to porosity, hydrophibility as well as the thermal behaviours, meanwhile the relationship between material composition and macroscopical performance of scaffolds was elucidated. Then, we studied how the degradation rates and mechanical properties of the scaffolds could be fine-tailored by adjusting the ratios, so as to satisfy some specific requirements for the desired tissue. Lastly, we evaluated the biological characteristics of scaffolds according to the results of cellular adhesion and proliferation. Taken together, these findings support our notion that PLLA/PLGA/HA composite scaffolds could be a lead candidate material for tissue engineering.
Porous scaffolds were fabricated by a novel method named high-pressure compression molding/salt leaching techniques. The self-made high-pressure compression molding apparatus is schematically shown in Fig. S1a,† meanwhile the corresponding temperature and pressure protocol are shown in Fig. S1b.† Fig. S2† illustrates the detailed experimental procedures about the fabrication of porous PLLA/PLGA/HA composite scaffolds as described in our previous paper.16
Porosity = (mNaCl/ρNaCl)/(mNaCl/ρNaCl + mPLLA/ρPLLA + mPLGA/ρPLGA + mHA/ρHA) × 100%, | (1) |
Connectivity = (m0 − m′)/m0NaCl × 100%, | (2) |
Density = m′/(mNaCl/ρNaCl + mPLLA/ρPLLA + mPLGA/ρPLGA + mHA/ρHA) × 100%, | (3) |
Morphology of the cells cultured on scaffolds was observed using SEM. Cells cultured for 3 days were fixed with 2.5% glutaraldehyde for 2 h at 4 °C. After they had been thoroughly washed with PBS, the samples were dehydrated sequentially through a series of increasing ethanol concentrations (10, 30, 50, 70, 80, 90, 95, and 100%) for 15 min × 2. Finally, scaffolds were dried overnight, coated with Au and examined with SEM. In addition, number of MC3T3-E1 cells was evaluated by adding Cell Counting Kit-8 (CCK-8, Dojindo, Japan) solution to each well. After 1, 3 and 7 days of incubation, numbers of live cells in the samples were counted by measuring the absorbance of the resulting medium at 450 nm through an ELISA reader (Model 550; Bio-Rad, Hercules, CA, U.S.A.). Absorbance at 600 nm was used for baseline correction.
Statistical analysis: all data were expressed as mean ± standard deviation, statistical software SPSS 13.0 was used to analyze the data by one-way analysis of variance; probability value of less than 0.05 was considered significantly different and that below 0.01 or 0.001 was considered highly significantly different.
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Fig. 1 SEM morphologies (a and b) and bimodal pore size distribution (c) and HA particles dispersion (d) of porous PLLA/PLGA/HA scaffolds. |
It can be seen from Fig. 1c that PLLA/PLGA/HA porous scaffolds are obtained with pores of average size between 100 and 150 μm. Zhou et al. once reported that, compared with scaffolds bearing smaller pores (10–50 μm), scaffolds with larger pores (>100 μm) allowed more deposition of collagen and provided enough space for cells vascularization.20 Of great interests, a bimodal porosity characterized by two different pore size ranges is observed in scaffolds. In detail, macropores (10–350 μm in diameter) represent the 74.4% of the total scaffold porosity with an average pore size of about 149.13 μm, while the remaining 25.6% are micropores (1–10 μm in diameter) with an average pore size of 7.65 μm.17 According to the literatures, it is deduced that the interconnected macropores are created by the leaching of continuous NaCl phase, while the closed micropores appeared in the walls of macropores are resulted from some small-sized NaCl droplets which remains as dispersed phase in the skeleton.21 In fact, such bimodal pore structure could be beneficial to the cell–cell communication and nutrient transportation within the scaffolds.22
In most approaches, bioactive materials (such as HA particles) are often deposited onto the surface of prepared porous scaffolds. Weak coating combination of the as-prepared scaffolds is the main disadvantage of these fabrication methods.23 HA particles are partially connected with the polymer matrix and mainly bonded with a weak strength of van der Waals's forces. Differently, combination of high-pressure molding and salt leaching method in current study produces a strict and regular architecture of scaffolds due to the melting-process with high pressure. On this context, HA particles herein were immersed and stayed tightly in PLLA/PLGA matrix by the pressure seepage, guaranteeing the scaffolds a highly improved biocompatibility (Fig. S3 and S4†). Furthermore, as shown in Fig. 1d, a homogenous distribution of HA particles in the scaffolds is revealed and no agglomeration is seen, indicative of an appropriate filler content.
Porosity and pore interconnectivity of scaffolds are critical for cell adhesion and proliferation, along with the diffusion of nutrients and oxygen. Values of porosity, interconnectivity and bulk density of scaffolds for each PLLA/PLGA ratio are measured and the results are shown in Table 1. As expected, at a constant mass fraction of NaCl, porosity of these scaffolds ranges from 81.5% to 82.7%, remaining almost invariable with the changes of PLLA/PLGA ratios. Porosity is often created in solid scaffold by the inclusion of porogen that can be leached away upon placement in an aqueous environment,24 thus mainly dependent on the content of porogen. It is worthy to be noticed that porosity of all scaffolds studied here constantly maintains in high level of above 80%, well meeting the requirements of application in tissue engineering and being able to provide enough space to guide cell proliferation.25 Similarly, variation of PLLA/PLGA ratios has no detectable influence on pore interconnectivity of the formed scaffolds. Its values nearly equalled to 98%, which demonstrates that NaCl within the mixtures is almost fully continuous. The highly interconnected porous materials can be easily seeded with a fluid phase containing cells, for it is desirable for permitting cell infiltration into and throughout the scaffolds. In terms of density obtained by weighing a sample of specific volume, it is just fluctuated around 0.25 g cm−3, nearly the lowest density reported so far for porous PLLA or PLGA scaffolds at such a high porosity and connectivity,10 fully embodying the scaffold prominent superiority of being lightweight. To support all these conclusions, Table 1 is performed in order to confirm the influence of PLLA/PLGA ratios on physical parameters.
PLLA/PLGA | Porosity (%) | Connectivity (%) | Density (g cm−3) | Tg (°C) | Tm (°C) | Water absorption rate (%) | Maximum load (N) | Compressive modulus (MPa) |
---|---|---|---|---|---|---|---|---|
a Note: Porosity, connectivity and the measured density of scaffolds were determined by gravimetric measurements according to eqn (1)–(3), separately. | ||||||||
10![]() ![]() |
82.7 | 97.7 | 0.250 | 64.1 | 174.4 | 166.7 ± 7.61 | 76.39 ± 2.4 | 6.48 ± 0.55 |
7![]() ![]() |
83.6 | 98.8 | 0.234 | 60.4 | 170.8 | 220.0 ± 10.12 | 54.08 ± 1.8 | 5.51 ± 0.14 |
5![]() ![]() |
81.7 | 97.3 | 0.262 | 61.0 | 170.3 | 257.1 ± 5.80 | 35.1 ± 2.1 | 4.64 ± 0.20 |
3![]() ![]() |
81.5 | 97.1 | 0.264 | 57.7 | 163.3 | 300.0 ± 10.25 | 18.81 ± 1.5 | 3.42 ± 0.12 |
0![]() ![]() |
81.8 | 97.8 | 0.259 | 56.8 | 152.8 | 357.2 ± 12.40 | 4.86 ± 0.6 | 1.12 ± 0.06 |
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Fig. 2 Trend of the % weight loss of PLLA/PLGA/HA scaffolds with different PLLA/PLGA weight ratios as a function of degradation time. Data were presented as mean ± standard deviation (n = 3). |
It is of great interest to notice that changes of foam weight loss are relatively gentle within the first 4 weeks, which have a tendency to exhibit more and more abruptly afterwards. A possible explanation of the observed behaviour stems from the theory of “stage degradation”.27 Scaffolds degradation roughly proceeds in two stages: first is a quasi-stable stage, where water absorption and plasticization occur together and cause a slight increase in the weight loss. Afterwards, along with the extension of immersion time in PBS, disruption of the pore structure and formation of blisters facilitate the degradation and bring about a stage of massive weight loss. In a word, combination of polymers with different degradation kinetics in such “high-pressure compression molding” way, is an effective approach to obtain scaffolds with controlled degradation rates according to some specific biomedical applications.
Water absorption is regarded as an important parameter that represents the efficiency of nutrient transfer within the scaffolds. Suitable water absorption ability is extremely necessary to obtain an ideal scaffold, for the high value of it will destroy the scaffolds shape. On the contrary, the terrible absorption will result in the lack of water and finally affect cells normal growth.28 Hydrophilic nature of materials is an important factor that influences the extent of scaffolds swelling capacity. As shown in Fig. 3, water absorption rate of the scaffolds is changed with ratios of PLLA/PLGA; it increases significantly from about 166.7 ± 7.61% to 357.2 ± 12.40% when the ratio varies from 10:
0 to 0
:
10. In comparison with PLLA, PLGA is a relatively hydrophilic material and therefore has a very high water uptake.29 Accordingly, the water contact angle measurement is utilized to further understand the hydrophilic and hydrophobic nature of different blend ratios. As shown in Fig. 3, the contact angle values are 71.04, 53.13, and 36.51° for scaffolds with PLLA/PLGA ratio of 10
:
0, 5
:
5, and 0
:
10, respectively. Along with the blending of PLGA in PLLA, an obvious decrease in contact angle value is presented, indicative of an improved hydrophilicity of scaffolds surface quality. Additionally, by and large, hydrophilic surfaces would give a contact angle between 0° and 90°.30 Each contact angle detected here is less than 75°, which in turn points towards the excellent hydrophilic behaviour of our fabricated PLLA/PLGA/HA composite scaffolds. Overall, there is a consistent pattern of such increased hydrophilicity with addition of PLGA, as observed from contact angle and water uptake results, which could impart scaffold degradation as well as cell attachment on it.
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Fig. 3 Water absorption rate and the contact angle images of PLLA/PLGA/HA scaffolds as a function of PLLA/PLGA ratios. Data were presented as mean ± standard deviation (n = 3). |
As we all know, scaffolds should have strength as close as possible to that of bone to be repaired or substituted, so that providing enough structural stability to support cell attachment. Compression tests are performed to assess the strength of our fabricated PLLA/PLGA/HA scaffolds. Fig. 5a and b illustrate the maximal load and compressive modulus of scaffolds, respectively, as a function of PLLA/PLGA ratios. With regard to the pure PLA/HA scaffolds, they display strong ability to resist deformation with the maximum load and compressive modulus high up to 76.39 ± 2.4 N and 6.48 ± 0.55 MPa, separately. Compared with the maximal value (21.7 N, 3.36 MPa) of PLA/HA scaffolds once reported with similar porosity and composition, it is at least two times higher, fully revealed an encouraging progress in developing load-bearing scaffolds. Conversely, the pure PLGA/HA scaffolds show the lowest compressive properties (4.86 ± 0.6 N, 1.12 ± 0.06 MPa). Mechanical integrities of composite scaffolds are intermediate between those of the pure components, which exhibit a trend of decline with the increase in mass fraction of PLGA.
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Fig. 5 Results of compression tests on composite scaffolds with various ratios of PLLA/PLGA. Data were presented as mean ± standard deviation (n = 3). |
In addition, it is interesting to observe that decrease of the mechanical properties is relatively gentle as PLGA content below 50%, which is on the order of 28.4% and 75.9% for scaffolds at PLLA/PLGA ratio of 5:
5 and 0
:
10, separately. As a result, it is reasonable to make a conclusion that incorporation of PLGA into PLLA weakens the carrying capacity of scaffolds. In order to obtain scaffolds with high resistance to external load, choice of PLGA content needs to be less than 50%. One side, mechanical properties of PLLA are more advantageous than those of PLGA, which tends not to deform under the presence of a compressive load for its stiffness.35 On the other side, it was once proposed that in the case of PLGA content less than 50%, the majority phase PLLA was crystalline and the minority PLGA was amorphous, therefore a dense structure and tight connection were provoked.17 On the whole, above data indicates that variation of PLLA/PLGA ratios do show significant influence on modifying mechanical properties of the composite scaffolds at a certain degree.
As above mentioned, changing PLLA/PLGA weight ratios in composite scaffolds is possible to obtain scaffolds with easily-tunable physical properties adapted to some specials needs of each application. Considering the requirements of scaffolds design on both biodegradability and mechanical performance aspects, the overall properties of porous composite scaffolds are undoubtedly optimal when the weight ratio of PLLA/PLGA is 5:
5. At first, the composite scaffolds experience 67.21 ± 3.14% weight loss after 12 weeks of incubation, which are not only complying with the degradation demands (over 65% in 12 weeks), but also maintaining the structure integrity during the long duration of tissue regeneration. Secondly, compressive modulus of the composite scaffolds reaches up to 4.64 ± 0.2 MPa, a quantity comparable to human cancellous bone (2–10 MPa).36 Such excellent mechanical property could provide a sufficient structural stability to support cell attachment, for which the scaffolds tend not to deform under the presence of a compressive load. Except for these, some other physical characteristics such as porosity of above 80% and contact angle less than 75° further verify the acquisition of a highly interconnected porous structure and a good hydrophilic behavior, which could impart the scaffold a good cell attachment on it. On the basis that the scaffolds are providing a sufficient mechanical strength to ensure the structural integrity, meanwhile satisfying the physiological criteria to guide tissue regeneration. In the following study, cell proliferation assay was performed using the composite scaffolds with PLLA/PLGA weight ratio of 5
:
5 to evaluate the toxicity of our fabricated resultants.
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Fig. 6 SEM micrographs of (a) PLLA/PLGA/HA scaffolds with PLLA/PLGA ratio of 5![]() ![]() |
Fig. 7 shows the relative cellular proliferation after cultured on cell culture plate and high-pressure fabricated PLLA/PLGA/HA composite scaffolds with PLLA/PLGA ratio of 5:
5 for 1, 3 and 7 days. In line with the result of cells morphology, cells number of the high-pressure compression molded PLLA/PLGA/HA composite scaffolds is matched to that of the plastic plate at all culturing time points, further suggesting that our fabricated scaffolds possess a great potential to support cellular proliferation. The morphology and proliferation of cells on the high-pressure compression molded scaffolds are further compared with those on the conventional compression molded scaffolds (as shown in Fig. S5†).
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06906a |
This journal is © The Royal Society of Chemistry 2016 |