Materials Science and Engineering C 38 (2014) 227–234
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
In vitro degradation of porous PLLA/pearl powder composite scaffolds Y.S. Liu a, Q.L. Huang a, A. Kienzle c, W.E.G. Müller c, Q.L. Feng a,b,⁎ a b c
State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Institut für Physiologische Chemie, University Medical Center, Johannes Gutenberg University Mainz, Universität, Duesbergweg 6, 55099 Mainz, Germany
a r t i c l e
i n f o
Article history: Received 22 October 2013 Received in revised form 30 December 2013 Accepted 4 February 2014 Available online 12 February 2014 Keywords: Degradation PLLA Aragonite Vaterite Composite scaffold
a b s t r a c t The in vitro degradation behavior of poly-L-lactide (PLLA), PLLA/aragonite pearl powder and PLLA/vaterite pearl powder scaffolds was investigated. The scaffolds were soaked in phosphate buffer solution (PBS) up to 200 days. Scanning electron microscopy (SEM), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) were used to observe any degradation of the scaffolds. Degradation behaviors such as changes in pH, porosity, bulk density, water absorption, weight loss and mechanical properties were discussed. The results show that a gradual increase of the pH in composite scaffolds can decrease the rate of hydrolysis of PLLA. PLLA/vaterite and PLLA/aragonite scaffolds have a similar degradation behavior but a slower rate of degradation than PLLA. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
1. Introduction Many biodegradable polymers with approved qualities by the USA Food and Drug Administration (FDA) have drawn attention for a possible use in tissue engineering due to their excellent performances. One of these predominant biodegradable polymers is the poly(L-lactide) PLLA [1,2]. PLLA exhibits beneficial properties including biocompatibility, biodegradation to innocuous lactic acid, an appropriate strength and modulus, as well as the capacity to improve cell attachment and proliferation [3–7]. Moreover, as PLLA can be easily prepared for 3D porous scaffolds in various shapes with suitable pore structures and porosity, it is therefore an attractive alternative for applications in tissue engineering [8,9]. PLLA-based porous scaffolds, modified in many aspects such as structures, shapes or surface [10,11], feature a series of properties for specific applications. Recently, much attention has been paid on a scaffold strategy for bone regeneration with osteoinductive factors such as BMP-2 [12]. Therefore, a PLLA-based scaffold with signal molecules that improves its osteoinductivity is looked forward to. A possible way to reach this is the addition of certain particles into PLLA to fabricate composite scaffolds. Such particles, including bioactive particles and pearl powder, have gained wide acceptance to enhance biological and mechanical performances of scaffolds [8,13,14]. Previous works proved that with the presence of bioactive particles the improved mechanical properties ⁎ Corresponding author at: Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China. Tel.: +86 10 62782770; fax: +86 10 62771160. E-mail address: [email protected] (Q.L. Feng).
http://dx.doi.org/10.1016/j.msec.2014.02.007 0928-4931/Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
of composite scaffold are derived from stiff particles binding into the soft polymer, also endowing bioactive behaviors to the polymer. These bioactive particles display the ability of promoting soft tissues to attach to hard connective tissues. Bioactive ceramics such as hydroxyapatite also exhibits the ability of bonding to hard tissues [14,15]. Pearl powder is biocompatible and biodegradable when implanted into bone defects [16]. Pearls contain growth factors that are able to increase osteoblast proliferation [17]. Aragonite and vaterite are two different calcium carbonate crystals [8,18]. Both, aragonite and vaterite are unstable crystals and easily transform into calcite crystals in solution [18]. Inorganic parts of lustrous pearls are aragonite crystals, which are called aragonite pearls. Aragonite pearl is composed of aragonite crystals and organic matrixes (b5 wt.%). Some pearls show no luster, containing pure vaterite crystals and around 5 wt.% organic matrixes, which are known as vaterite pearls [19,20]. Our previous works revealed that PLLA/aragonite composite scaffold showed better cytocompatibility, while PLLA/vaterite scaffold displayed lower proliferation and osteogenic differentiation rate of rat bone marrowderived mesenchymal stem cells (rBMSCs) than PLLA scaffold [21]. Materials formulated as scaffolds for tissue engineering applications have to meet a range of requirements including biocompatibility, degradation rate, mechanical performances and non-toxicity [22]. Degradation behaviors including degradation rate and property change of scaffolds in a short or long-term period are very significant, as they possibly affect cell behaviors, such as cell attachment, proliferation, differentiation and the expression of cell functions. Furthermore it might influence tissue regeneration because scaffolds and degradation products can offer osteoconductive and osteoinductive microenvironments [23]. The ideal degradation rate is a balance between scaffold
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degradation and tissue regeneration [2]. For the application of a scaffold in tissue engineering, it is important to know its degradation rate in vitro in order to predict its in vivo degradation behavior. In vitro degradation of PLLA scaffold and its blends has been widely investigated [24,25]. Crystallinity, molecular weight and hydrolytic action mainly determine the degradation rate of PLLA [2]. During degradation oligomers of low molecular weight PLLA are formed by an intramolecular reaction [26]. Autocatalysis plays a major role in the processes of degradation. The formation of acidic products and acidic chain ends could speed up ester fracture and therefore increases the PLLA degradation rate [2,27]. Some degradation behaviors of polymers may be influenced by particles added in composite scaffolds. The addition of hydroxyapatite particles into PLLA scaffold slows down the decrease of pH and significantly reduces the weight loss [28]. Moreover, bioglass particles could decrease the thermal stability of the polymers by acting as a catalyst [14,29]. Pearl powder exhibits suitable biodegradability and biocompatibility. However, there are only few reports on pearl powder applications in bone regeneration. We have developed a novel PLLA/aragonite scaffold and PLLA/vaterite scaffold recently [8]. It is necessary to further evaluate their physicochemical and biological properties. This work focuses on the performances of PLLA/aragonite scaffold and PLLA/vaterite scaffold in the processes of degradation, such as water absorption, weight loss, mechanical properties, molecular weight and crystallinity. These properties are helpful to predict the biological properties of composite scaffolds in vivo. 2. Materials and methods 2.1. Materials PLLA (Mw = 17.9 × 104 Da) was purchased from Shandong Medical Device Company. The 1,4-dioxane was purchased from Beijing Modern Eastern Fine Chemical Co., Ltd. 99.7% ethanol was purchased from Beijing Chemical Works. All the reagents were of analytical grade. Fresh water pearls were obtained from freshwater cultured Hyriopsis cumingii in Zhejiang, China. 2.2. Preparation of samples 2.2.1. Pearl powder preparation Pearls were collected from freshwater cultured H. cumingii. Vaterite and aragonite pearls were acquired from selected lustrous pearls and lack luster pearls respectively [8]. The two specimens were put into a beaker filled with ethanol at ambient temperature for 10 h, then rinsed in ultra-pure water (Milli-Q) and air dried. The pearls were grounded into powder using a grinder with a maximum speed of 25,000 rpm for 3 min. Most of the powders in large size are in the container of the grinder, while a small amount of powders in small size are sticked to the surface of the lid of the grinder. Only the powders sticked to the surface of the lid are scraped and obtained. The fine powder was obtained by passing it through a sieve with a pore diameter of 50 μm. The mean size of the used powder is 17.5 μm.
2.3. In vitro degradation The PLLA, PLLA/aragonite and PLLA/vaterite scaffolds were cut into regular cylindrical shape, 9 mm in diameter and 18 mm in length. The amount of fine pearl powder in phosphate buffer solution (PBS) is as high as in composite scaffolds. The degradation was evaluated for up to 200 days. Before testing the long-term degradation, all scaffolds were pretreated as follows to guarantee that PBS could access all pores of the scaffolds: the scaffolds were placed into a beaker filled with PBS solution which was covered by a plastic wrap with some little holes in it. The beaker was put into a vacuum freeze-drier. By repeating this process of vacuum/air for several times, the scaffolds sank in the solution and it is guaranteed that PBS could enter all pores of the scaffolds. Each scaffold and fine pearl powder was put in individual centrifuge tubes with 15 ml PBS in a shaker working at 37 °C with 60 rpm. The initial pH of PBS is 7.40. The medium was replaced by fresh PBS every week except for pH and concentration of Ca2+ groups. For pH groups, the PBS solution was not changed. For concentration of Ca2 + groups, fifteen samples of each scaffold and pearl powder were prepared at five designated points of time (1, 7, 14, 28, 42 days) and the PBS solution was not changed. 2.4. Characterizations 2.4.1. pH and concentration of Ca2+ The pH value of PBS containing the scaffolds or pearl powders was measured using a pH meter (FE20K, Mettler Toledo, Switzerland) at designated points of time. 1 ml PBS solution was acquired from the solutions containing the scaffolds or pearl powders to measure the concentration of Ca2+ using HITACHI-7170 automated clinical analyzer. 2.4.2. Gel permeation chromatography (GPC) Molecular weight and molecular weight distribution of the PLLA were estimated using gel permeation chromatography (GPC, Waters, USA). A Waters 1515 GPC with a Waters 2414 differential refractive index detector was operated at 25 °C. Chloroform was used as the mobile phase with a flow rate of 1.0 ml/min. About 90 mg scaffolds were dissolved in 10 ml chloroform and subsequently filtered through a 0.45 μm syringe filter before testing. Polystyrene was used as the standard solution for calibration in a broad range of 0.5–1000.0 kDa. 2.4.3. Differential scanning calorimetry (DSC) To measure glass transition temperature and melting temperature of the composites, DSC was carried out with a TA DSC instrument (DSC Q2000, TA, America). The samples before and after degradation were heated from 0 °C to 210 °C at a speed of 10 °C/min. Afterwards, the samples were quenched and a reheating scan was carried out from 0 °C to 210 °C at a speed of 10 °C/min to get rid of thermal history and to determine the thermal transition of the scaffolds. The crystallinity degree (Xc) of the scaffolds was calculated according to the following Eq. (1) [2]:
Xc ¼ 2.2.2. Scaffold preparation Scaffolds were prepared by freeze-drying method [8]. PLLA was dissolved in 1,4-dioxane to obtain a 5% m/v PLLA/1, 4-dioxane solution. The fine aragonite powders were added to the PLLA solution resulting in a final polymer/powder weight ratio of 80/20. The solution was stirred for 15 min and sonicated for 30 min. Then the solution was transferred into a cylindrical mold and frozen at − 20 °C for 24 h. The cylindrical molds were lyophilized at −60 °C for 48 h. The PLLA/vaterite scaffold was fabricated the same way. The PLLA scaffold was prepared the same way without powder. Before tests, all scaffolds were dried in a vacuum oven for 3 days to get rid of the residual 1,4-dioxane.
ΔH m 100% ΔH ref
ð1Þ
where, ΔHm is the melting enthalpy for crystals of PLLA in the scaffolds, ΔHref is the melting enthalpy for pure crystals of PLLA. ΔHref = 90.95 J/g. 2.4.4. Water absorption and weight loss At each point of time, three samples of each scaffold were taken out of the PBS, wiped off the surface solution with filter paper and weighed to determine the wet weight (Ww). These samples were washed in ultra-pure water (Milli-Q), dried in a vacuum oven for 3 days and weighed to obtain the constant weight (Wd). Water absorption and
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weight loss of the scaffolds were calculated according to the following equations: Water absorption ¼
Weight loss ¼
W w −W d 100% Wd
W i −W d 100% Wi
ð2Þ
ð3Þ
where Wi is the original dry weight of the scaffold. 2.4.5. The mechanical properties The compressive strength and modulus of the composite scaffolds before and after PBS immersion were tested using a Zwick Z005 universal testing machine at ambient temperature. The samples were compressed at a rate of 1 mm/min until the strain was N 30%. The compressive strength was calculated by the maximum value of the linear part of the stress–strain curve [8]. The compressive modulus was determined by the slope of the initial linear region of the curve. Three samples for each scaffold were tested. By averaging the intermediate data the final result was determined. 2.4.6. SEM The bulk samples before and after degradation were dried in vacuum and fractured. The fractured morphology of scaffolds was observed using a scanning electron microscopy (SEM, FEI Quanta 200) at 30 KV after sputter coating with gold. 2.4.7. Mercury intrusion porosimetry The density and porosity of the scaffolds were assessed by a mercury intrusion analyzer (Autopore IV 9510, Micromeritics, America). All samples were weighed and kept in a cell under vacuum condition. At such lower pressure the pores of the samples cannot be intruded by mercury. Increasing pressures raised the amount of mercury entering the pores of the samples. The pressure of mercury was inversely proportional to the pore volume of the materials. The density was subsequently calculated by the weight and volume of the samples. The porosity was calculated according to the following Eq. (4) [30]: P l −P g ¼
4σ cosθ d
ð4Þ
where Pl is the pressure of mercury (Pa), Pg is the pressure of the gas (Pa), σ is the surface tension of mercury, θ is the contact angle and d is the pore diameter.
Fig. 1. pH of PBS containing the scaffolds or pearl powders. * shows significant differences between groups (p b 0.05). ** indicates significant differences between groups (p b 0.01).
Because vaterite is more easily dissolved than aragonite, PLLA/vaterite scaffold releases more alkalis into the PBS than PLLA/aragonite scaffold. The dissolution of CaCO3 from vaterite and aragonite produces an alkalinity in PBS, which could neutralize acid residues from PLLA and slow down the hydrolysis rate of PLLA [31]. 3.2. Concentration of Ca2+ in PBS solutions containing scaffolds or pearl powders Fig. 2 shows changes of the Ca2+ concentration in PBS containing the scaffolds or pearl powders. PBS and PLLA scaffolds do not release Ca2+. The concentration of Ca2 + in PBS containing vaterite powders is 0.13 mM on day 1 and increases slowly for 42 days, while in PBS containing aragonite powders, the initial Ca2+ concentration is 0.06 mM on day 1 and gradually rises to 0.20 mM within 28 days. As vaterite is more easily dissolved than aragonite, vaterite could release more Ca2+ and rapidly reach the high Ca2 + concentration on the first day. It is shown that the Ca2+ concentration is different for these two powders.
3. Results and discussion 3.1. pH of PBS containing scaffolds or pearl powders To investigate the degradation of the scaffolds and the pearl powders, pH changes of the solutions are measured (Fig. 1). The pH of PLLA stays at 7.40 in the first 20 days. By the 21st day a small observable decrease in pH occurs. The pH of PLLA remains constant below 7.40 during the degradation time. PLLA starts to degrade in PBS causing a change of pH. In contrast, pH of vaterite and aragonite powders rapidly increases to 7.80 and 7.65 on the 10th day respectively. The maximum pH value of vaterite powder is 8.60, remaining high all through the degradation time. Compared to pure PLLA scaffold and pearl powders, pH of PBS with composite scaffolds shows a different trend. In both composite scaffolds, the pH is higher than 7.40 and remained around 7.80 during the first 100 days. The dissolution of CaCO3 in composite scaffolds causes the gradual increase of pH. The degradation solutions containing PLLA/vaterite scaffold show a similar change of the pH value like PLLA/aragonite scaffold, but the pH of PLLA/vaterite scaffold keeps consistently higher than the pH of the PLLA/aragonite scaffold.
Fig. 2. Concentration of Ca2+ in PBS containing the scaffolds or pearl powders.
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Table 1 Molecular weight and molecular weight distribution of PLLA before and after degradation. Degradation time (days)
Scaffolds
Mw (Da)
0 100 100 100 200 200 200
PLLA PLLA PLLA/vaterite PLLA/aragonite PLLA PLLA/vaterite PLLA/aragonite
17.9 15.5 15.6 16.6 14.6 15.6 14.8
× × × × × × ×
104 104 104 104 104 104 104
Mn (Da) 12.7 8.1 9.0 9.5 2.8 2.5 3.4
× × × × × × ×
104 104 104 104 104 104 104
The two kinds of pearl powder show a similar size distribution from 0–23 μm (data not shown). Pearl powder easily aggregates to form small or large conglomerates in PBS, possibly affecting the dissolution of CaCO3. Composite scaffolds have a greater increase in releasing Ca2 +on day 7 and increase slowly after 7 days. Powders are more homogeneously dispersed in scaffolds than in PBS, therefore, composite scaffolds release more Ca2+ than pearl powders all through the degradation time. PLLA/ aragonite scaffold shows a similar change of the Ca2+ concentrations as PLLA/vaterite scaffold. Because vaterite is more easily dissolved than aragonite, Ca2+ concentrations of PLLA/vaterite scaffold keep consistently higher than of PLLA/aragonite scaffold. Buffer solutions are used to keep pH. PBS solution containing HPO2− 4 2+ and H2PO− concentration at a nearly con4 has abilities to stabilize Ca stant value. Previous works have studied the solubility of calcite and aragonite in seawater at 35‰ salinities, 25 °C and one atmosphere [32]. Calcite, aragonite and vaterite are three different CaCO3 crystals. Ksp values of calcite and aragonite are 10−6.37and 10−6.19 respectively. Ca2 + concentrations for calcite and aragonite could reach 0.653 mM and 0.804 mM. Because vaterite is easier to dissolve than aragonite, it is reasonable for higher Ca2+ concentrations of vaterite than aragonite. Thus, Ca2 + concentrations for composite scaffolds do not reach the saturation level for 42 days and just increase slowly by a buffering effect.
Mw/Mn
Peak of molecular weight (Da)
1.407 1.910 1.734 1.736 5.088 6.275 4.273
17.4 17.2 17.2 17.2 16.4 16.6 16.4
× × × × × × ×
104 104 104 104 104 104 104
Other peaks of molecular weight (Da)
1.1 × 104 1.0 × 104 1.1 × 104
scaffold corresponding to 1.0 × 104 or 1.1 × 104 Da. Meaning that some water-soluble PLLA oligomers may be separated from the scaffolds. PLLA in composite scaffold shows a relatively slower decrease of molecular weight compared with the pure PLLA scaffold. After 200 days molecular weight of PLLA in PLLA scaffold reduced from 17.9 × 104 Da to 14.6 × 104 Da, while in composite scaffold, it reduced to 15.6 × 104 in PLLA/vaterite and to 14.8 × 104 in PLLA/aragonite. It can be seen that compared to the PLLA scaffold, PLLA in composite degraded at a slower rate. The result is consistent with the pH change, meaning that alkalinity products from powders could provide a relatively steady environment decreasing the speed of PLLA degradation. The addition of powders into PLLA could decrease PLLA degradation rate. PLLA/vaterite scaffold shows a higher molecular weight of PLLA compared to PLLA/ aragonite scaffold. This may be highly related to the fact that more alkalinity products are released from vaterite powders than from aragonite
3.3. Molecular weight Changes of the molecular weight of PLLA in scaffolds during the degradation process are shown in Table 1. In general, all molecular weights of PLLA in scaffolds decrease during the degradation process. The molecular weight distribution (Mw/Mn) of PLLA in all scaffolds increases, which means a decrease in high molecular weight, with a subsequent increase in low molecular weight of PLLA. The peak of molecular weight of PLLA moved towards the lower molecular weight. PLLA in pure PLLA scaffold shows a dramatic decrease of molecular weight and a rapid increase of the molecular weight distribution (from 1.407 to 5.088). The chains fractured from its backbone and hydrolysis resulted in a decrease in molecular weight of PLLA. It often appears in the amorphous phase of PLLA [33]. It was reported that PLLA oligomers with a molecular weight below 104 Da are partially hydrosoluble [34]. On the 200th day there is another peak corresponding to 1.1 × 104 Da in the PLLA scaffolds. In composite scaffold, the low molecular weight PLLA also separated from PLLA Table 2 Characteristic thermal transition temperature of the scaffold before and after degradation according to the reheating scan. Scaffolds
Tg (°C)
Tm (°C)
ΔHm (J/g)
Crystallinity
PLLA PLLA/vaterite PLLA/aragonite PLLA 200 days PLLA/vaterite 200 days PLLA/aragonite 200 days
58.46 58.26 59.19 59.56 60.84 60.50
173.73 181.08 178.32 173.51 174.96 174.17
35.93 32.88 33.27 47.80 34.21 35.02
39.50% 36.15% 36.58% 52.55% 37.60% 38.50%
Fig. 3. DSC curves of the scaffolds before (a) and after 200 days degradation (b).
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Fig. 4. Water absorption of the scaffolds against degradation time.
powders. Thus, it could provide a steadier environment for declining PLLA degradation. 3.4. Differential scanning calorimetry (DSC) Table 2 shows that PLLA in PLLA scaffold has a glass transition temperature (Tg) of 58.46 °C and a melting temperature (Tm) of 173.73 °C, slightly differing from what can be found in literature [14]. After 200 days of degradation, Tg increased to 59.56 °C and Tm decreased to 173.51 °C. Fig. 3 also shows that the shape of the melting peak remains similar before and after degradation for PLLA scaffold, which indicates that there is no significant change in PLLA crystals after 200 days of degradation. With the addition of pearl powder into PLLA Tg decreases and Tm increases compared to PLLA. After 200 days of degradation Tg increases and Tm decreases in both composite scaffolds. Also, the shapes of melting peaks are changed. These results show that in composite scaffolds PLLA polymer has lower mobility after degradation and the PLLA crystals undergo some changes during degradation. The partially degraded main chains of PLLA may be the main factor for those characteristic temperature changes [14]. For two composite scaffolds, there are differences between Tg, Tm and the shape of the melting peak, which
Fig. 6. Change of the compressive strength (a) and compressive modulus (b) of the scaffolds before and after degradation. ** indicates significant differences between groups (p b 0.01). * shows significant differences between groups (p b 0.05).
may arise from different molecular weight, crystallinity, etc. Table 2 also shows the crystallinity degree of the scaffolds. For PLLA scaffold, there is an increase of crystallinity from 39.50% to 52.55% during the 200 days of degradation. PLLA crystallinity can be increased by the major way [2]: the amorphous PLLA would be the first disappearing in solution in the process of hydrolysis. Composite scaffolds display lower crystallinities compared to PLLA after the 200 days of degradation. The autocatalytic degradation of PLLA usually combines with acidic products, which could be buffered by the release of alkalinity products from powders [35]. As the molecular weight of PLLA in composite scaffold is higher than in PLLA scaffold (Table 1), alkalinity products from powders could provide a relatively steady environment decreasing hydrolysis of the amorphous phase of PLLA. 3.5. Water absorption
Fig. 5. Weight loss of the scaffolds against degradation time. ** indicates significant differences between groups (p b 0.01).
Fig. 4 shows the water absorption of the scaffolds. All scaffolds show similar water absorption during the incubation. In the first 3 weeks, water absorption of the scaffolds quickly increases with a maximum of about 900%. After 3 weeks, the water absorption of the scaffolds stabilizes at the saturation points. Although powders and PLLA are hydrophobic materials, the water absorption of scaffold gradually increases. Literatures have reported that a decrease in sample mass
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a
b
c
d
e
f
g
h
i
j
k
l
PLLA scaffold
PLLA/vaterite scaffold
PLLA/aragonite scaffold
Fig. 7. SEM images of fractured morphology of the scaffolds before and after degradation. (a, d, g, j) PLLA scaffold, (b, e, h, k) PLLA/vaterite scaffold, (c, f, i, l) PLLA/aragonite scaffold. The scale bar is 100 μm.
and an increase in sample dimensions will cause a rapid increase in water absorption [36]. The high porosity (N88%) and large pore size (about 100 μm) allow water to enter into scaffold more easily (data not shown). For PLLA scaffold, during the first period of degradation, PLLA absorbing PBS to some extent, leading in swelling and hydrolytic degradation of low molecular weight PLLA, and the increased defects and porosity will gradually increase water absorption. The main water-binding ability of composite scaffold is corresponding to PLLA swelling and the dissolution of pearl powder, which provide more room for water. This rapid increase of water absorption also causes the high weight loss of the scaffold and the possible porous structure destruction [31].
3.6. Weight loss Fig. 5 shows the weight loss of the scaffolds in PBS. For PLLA scaffold, with increasing degradation time, the weight loss is not changing linearly and is showing four steps. There is no weight loss of pure PLLA scaffold in the initial stage. After 2–4 weeks, a rapid increase of weight loss to 3% can be observed. Afterwards it tends to a steady condition during week 4 to 14. After 14 weeks another rapid weight loss occurs. PLLA is a semicrystalline polymer. Side chains fractured from PLLA backbone could reduce the PLLA weight during 2–4 weeks. However, the backbone keeps steady during week 4 to 14 and starts to fracture again after 14 weeks. At the same time, the hydrolysis of low molecular
weight PLLA from PLLA porous scaffold may be attributed to the change of the weight loss. The weight loss of the composite scaffolds is relatively faster compared to the PLLA scaffold. During 1 to 4 weeks the composite scaffolds show a higher weight loss, slowly increasing after 4 weeks. Considering the fast decrease in the mechanical properties of composite scaffolds (Fig. 6) and gradual increase of pH in PBS (Fig. 1), the dissolution of CaCO3 from pearl powders is the main factor for the weight loss of the composite scaffolds in the first 4 weeks. After 4 weeks there is a complex process of degradation because at the same time PLLA possibly degrades and pearl powder dissolves. Alkalinity products released from the dissolution of CaCO3 could decrease the process of hydrolysis and slow down the PLLA degradation rate [29]. Because vaterite is more easily dissolved than aragonite, it is reasonable for a higher weight loss of PLLA/vaterite than PLLA/aragonite in the first 3 weeks. After 3 weeks both composite scaffolds show similar weight loss while PLLA starts to degrade. 3.7. The mechanical properties The mechanical properties of the scaffolds were measured by compressive measurements. The compressive strength and compressive modulus of the three scaffolds show a different decreasing trend (Fig. 6). For the PLLA scaffold, the compressive strength and compressive modulus significantly decreased during the first 30 days but much slower afterwards. In the first 30 days, the hydrolysis of PLLA of
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structure. In conclusion, to destroy the structure of the PLLA scaffolds and composite scaffolds, 200 days is not long enough. 3.9. Porosity and bulk density As expected, bulk density decreases and porosity increases with proceeding degradation time (Fig. 8). Before degradation, the addition of pearl powder into PLLA increased the density of the scaffold (Fig. 8(a)). Such high density can improve the mechanical properties of the scaffolds [37]. PLLA scaffold shows a dramatic decrease in density after the degradation. The hydrolysis of low molecular weight PLLA from PLLA porous scaffold, which may be an amorphous phase, contributes to the decrease of density. Because the mechanical properties of PLLA scaffold are mainly derived from both its porous structure and density, the observed change of density does not match the changes of the mechanical properties of the scaffolds after 30 days. In contrary, the composite scaffold shows a continuous decrease in density at a relatively slow speed, which is mainly caused by the dissolution of the pearl powder. For PLLA/vaterite scaffold the porosity increased from 88% to 97% after 200 days degradation (Fig. 8(b)). In contrast, for PLLA/aragonite and PLLA scaffold porosity reaches a maximum after 100 days and is decreasing afterwards again. The dissolution of the powder and the hydrolysis of PLLA increased the porosity. However, the relation of the appearance and disappearance of pores in the porous scaffold should be considered. The dissolution of pearl powder will increase porosity, while collapsing pores decrease porosity. Alkalinity products released from PLLA/vaterite scaffold could drop the PLLA degradation rate even more. The decrease of the amount of hydrolysis of PLLA will decline the number of collapsed pores. At the same time, since vaterite is more unstable and more easily dissolved than aragonite and PLLA, the dissolution of vaterite powders in scaffolds could cause a steady increase of porosity for up to 200 days. For the PLLA/aragonite scaffold, the balance between the dissolution of aragonite powder and collapsed pores may determine the final porosity of the scaffold. 4. Conclusions Fig. 8. Bulk density (a) and porosity (b) of different scaffolds during degradation. * shows significant differences between groups (p b 0.05).
low molecular weight from PLLA porous scaffold contributes to the decrease of compressive strength and compressive modulus of PLLA scaffold. After 30 days, the slight decrease of compressive strength and compressive modulus is derived from a steady porous structure of PLLA. The composite scaffolds display a gradual decrease of the compressive strength and compressive modulus. After 200 days, the compressive strength is still higher than that of the PLLA scaffold before degradation. The compressive modulus is slightly less than that of the PLLA scaffold before degradation. During the 200 days of degradation, the dissolution of pearl powders may be the main factor for a decreasing compressive strength and compressive modulus. At the same time, the dissolution of pearl powders and the hydrolysis of PLLA increasing defects will result in losing the order of whole porous structure and decreasing the mechanical properties of the scaffolds. 3.8. SEM Fig. 7 shows that there are some little holes appearing on the surface of the pore walls after degradation. With ongoing time, surface roughness of the pore walls increased in all scaffolds. For the PLLA scaffolds, the hydrolysis of PLLA and the recrystallization of partially degraded short chains of PLLA may have increased the surface roughness [2]. For composite scaffolds, the partial dissolution of pearl powders, the degradation of PLLA, and recrystallization onto the surface cause this roughness and irregular morphology. All samples maintain their porous
In this work, different degradation behaviors of PLLA, PLLA/vaterite and PLLA/aragonite scaffolds in PBS have been studied. The gradual increase of pH in composite scaffolds can decrease the hydrolysis rate of PLLA. Moreover, composite scaffolds show a greater ability to release Ca 2 + than pearl powders in PBS. During the degradation, PLLA cooperating with pearl powders displays changes such as mechanical properties, weight loss, bulk density and porosity. Alkalinity products released from composite scaffolds can provide a relatively steady environment for slowing down the degradation rate of PLLA and prolonging the degradation time of composite scaffolds. PLLA/vaterite and PLLA/ aragonite scaffolds show similar degradation behaviors. Further investigation could be performed on biological properties including degradation behavior in vivo and biocompatibility in order to promote the novel material in clinical applications. Acknowledgments The authors are grateful for the financial support from the National Natural Science Foundation of China (51361130032) and the Doctor Subject Foundation of the Ministry of Education of China (20120002130002). References [1] K. Madhavan Nampoothiri, N. Rajendran Nair, R. Pappy John, An overview of the recent developments in polylactide (PLA) research, Bioresour. Technol. 101 (2010) 8493–8501.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
In vitro degradation of porous PLLA/pearl powder composite scaffolds Y.S. Liu a, Q.L. Huang a, A. Kienzle c, W.E.G. Müller c, Q.L. Feng a,b,⁎ a b c
State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Institut für Physiologische Chemie, University Medical Center, Johannes Gutenberg University Mainz, Universität, Duesbergweg 6, 55099 Mainz, Germany
a r t i c l e
i n f o
Article history: Received 22 October 2013 Received in revised form 30 December 2013 Accepted 4 February 2014 Available online 12 February 2014 Keywords: Degradation PLLA Aragonite Vaterite Composite scaffold
a b s t r a c t The in vitro degradation behavior of poly-L-lactide (PLLA), PLLA/aragonite pearl powder and PLLA/vaterite pearl powder scaffolds was investigated. The scaffolds were soaked in phosphate buffer solution (PBS) up to 200 days. Scanning electron microscopy (SEM), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) were used to observe any degradation of the scaffolds. Degradation behaviors such as changes in pH, porosity, bulk density, water absorption, weight loss and mechanical properties were discussed. The results show that a gradual increase of the pH in composite scaffolds can decrease the rate of hydrolysis of PLLA. PLLA/vaterite and PLLA/aragonite scaffolds have a similar degradation behavior but a slower rate of degradation than PLLA. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
1. Introduction Many biodegradable polymers with approved qualities by the USA Food and Drug Administration (FDA) have drawn attention for a possible use in tissue engineering due to their excellent performances. One of these predominant biodegradable polymers is the poly(L-lactide) PLLA [1,2]. PLLA exhibits beneficial properties including biocompatibility, biodegradation to innocuous lactic acid, an appropriate strength and modulus, as well as the capacity to improve cell attachment and proliferation [3–7]. Moreover, as PLLA can be easily prepared for 3D porous scaffolds in various shapes with suitable pore structures and porosity, it is therefore an attractive alternative for applications in tissue engineering [8,9]. PLLA-based porous scaffolds, modified in many aspects such as structures, shapes or surface [10,11], feature a series of properties for specific applications. Recently, much attention has been paid on a scaffold strategy for bone regeneration with osteoinductive factors such as BMP-2 [12]. Therefore, a PLLA-based scaffold with signal molecules that improves its osteoinductivity is looked forward to. A possible way to reach this is the addition of certain particles into PLLA to fabricate composite scaffolds. Such particles, including bioactive particles and pearl powder, have gained wide acceptance to enhance biological and mechanical performances of scaffolds [8,13,14]. Previous works proved that with the presence of bioactive particles the improved mechanical properties ⁎ Corresponding author at: Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China. Tel.: +86 10 62782770; fax: +86 10 62771160. E-mail address: [email protected] (Q.L. Feng).
http://dx.doi.org/10.1016/j.msec.2014.02.007 0928-4931/Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
of composite scaffold are derived from stiff particles binding into the soft polymer, also endowing bioactive behaviors to the polymer. These bioactive particles display the ability of promoting soft tissues to attach to hard connective tissues. Bioactive ceramics such as hydroxyapatite also exhibits the ability of bonding to hard tissues [14,15]. Pearl powder is biocompatible and biodegradable when implanted into bone defects [16]. Pearls contain growth factors that are able to increase osteoblast proliferation [17]. Aragonite and vaterite are two different calcium carbonate crystals [8,18]. Both, aragonite and vaterite are unstable crystals and easily transform into calcite crystals in solution [18]. Inorganic parts of lustrous pearls are aragonite crystals, which are called aragonite pearls. Aragonite pearl is composed of aragonite crystals and organic matrixes (b5 wt.%). Some pearls show no luster, containing pure vaterite crystals and around 5 wt.% organic matrixes, which are known as vaterite pearls [19,20]. Our previous works revealed that PLLA/aragonite composite scaffold showed better cytocompatibility, while PLLA/vaterite scaffold displayed lower proliferation and osteogenic differentiation rate of rat bone marrowderived mesenchymal stem cells (rBMSCs) than PLLA scaffold [21]. Materials formulated as scaffolds for tissue engineering applications have to meet a range of requirements including biocompatibility, degradation rate, mechanical performances and non-toxicity [22]. Degradation behaviors including degradation rate and property change of scaffolds in a short or long-term period are very significant, as they possibly affect cell behaviors, such as cell attachment, proliferation, differentiation and the expression of cell functions. Furthermore it might influence tissue regeneration because scaffolds and degradation products can offer osteoconductive and osteoinductive microenvironments [23]. The ideal degradation rate is a balance between scaffold
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degradation and tissue regeneration [2]. For the application of a scaffold in tissue engineering, it is important to know its degradation rate in vitro in order to predict its in vivo degradation behavior. In vitro degradation of PLLA scaffold and its blends has been widely investigated [24,25]. Crystallinity, molecular weight and hydrolytic action mainly determine the degradation rate of PLLA [2]. During degradation oligomers of low molecular weight PLLA are formed by an intramolecular reaction [26]. Autocatalysis plays a major role in the processes of degradation. The formation of acidic products and acidic chain ends could speed up ester fracture and therefore increases the PLLA degradation rate [2,27]. Some degradation behaviors of polymers may be influenced by particles added in composite scaffolds. The addition of hydroxyapatite particles into PLLA scaffold slows down the decrease of pH and significantly reduces the weight loss [28]. Moreover, bioglass particles could decrease the thermal stability of the polymers by acting as a catalyst [14,29]. Pearl powder exhibits suitable biodegradability and biocompatibility. However, there are only few reports on pearl powder applications in bone regeneration. We have developed a novel PLLA/aragonite scaffold and PLLA/vaterite scaffold recently [8]. It is necessary to further evaluate their physicochemical and biological properties. This work focuses on the performances of PLLA/aragonite scaffold and PLLA/vaterite scaffold in the processes of degradation, such as water absorption, weight loss, mechanical properties, molecular weight and crystallinity. These properties are helpful to predict the biological properties of composite scaffolds in vivo. 2. Materials and methods 2.1. Materials PLLA (Mw = 17.9 × 104 Da) was purchased from Shandong Medical Device Company. The 1,4-dioxane was purchased from Beijing Modern Eastern Fine Chemical Co., Ltd. 99.7% ethanol was purchased from Beijing Chemical Works. All the reagents were of analytical grade. Fresh water pearls were obtained from freshwater cultured Hyriopsis cumingii in Zhejiang, China. 2.2. Preparation of samples 2.2.1. Pearl powder preparation Pearls were collected from freshwater cultured H. cumingii. Vaterite and aragonite pearls were acquired from selected lustrous pearls and lack luster pearls respectively [8]. The two specimens were put into a beaker filled with ethanol at ambient temperature for 10 h, then rinsed in ultra-pure water (Milli-Q) and air dried. The pearls were grounded into powder using a grinder with a maximum speed of 25,000 rpm for 3 min. Most of the powders in large size are in the container of the grinder, while a small amount of powders in small size are sticked to the surface of the lid of the grinder. Only the powders sticked to the surface of the lid are scraped and obtained. The fine powder was obtained by passing it through a sieve with a pore diameter of 50 μm. The mean size of the used powder is 17.5 μm.
2.3. In vitro degradation The PLLA, PLLA/aragonite and PLLA/vaterite scaffolds were cut into regular cylindrical shape, 9 mm in diameter and 18 mm in length. The amount of fine pearl powder in phosphate buffer solution (PBS) is as high as in composite scaffolds. The degradation was evaluated for up to 200 days. Before testing the long-term degradation, all scaffolds were pretreated as follows to guarantee that PBS could access all pores of the scaffolds: the scaffolds were placed into a beaker filled with PBS solution which was covered by a plastic wrap with some little holes in it. The beaker was put into a vacuum freeze-drier. By repeating this process of vacuum/air for several times, the scaffolds sank in the solution and it is guaranteed that PBS could enter all pores of the scaffolds. Each scaffold and fine pearl powder was put in individual centrifuge tubes with 15 ml PBS in a shaker working at 37 °C with 60 rpm. The initial pH of PBS is 7.40. The medium was replaced by fresh PBS every week except for pH and concentration of Ca2+ groups. For pH groups, the PBS solution was not changed. For concentration of Ca2 + groups, fifteen samples of each scaffold and pearl powder were prepared at five designated points of time (1, 7, 14, 28, 42 days) and the PBS solution was not changed. 2.4. Characterizations 2.4.1. pH and concentration of Ca2+ The pH value of PBS containing the scaffolds or pearl powders was measured using a pH meter (FE20K, Mettler Toledo, Switzerland) at designated points of time. 1 ml PBS solution was acquired from the solutions containing the scaffolds or pearl powders to measure the concentration of Ca2+ using HITACHI-7170 automated clinical analyzer. 2.4.2. Gel permeation chromatography (GPC) Molecular weight and molecular weight distribution of the PLLA were estimated using gel permeation chromatography (GPC, Waters, USA). A Waters 1515 GPC with a Waters 2414 differential refractive index detector was operated at 25 °C. Chloroform was used as the mobile phase with a flow rate of 1.0 ml/min. About 90 mg scaffolds were dissolved in 10 ml chloroform and subsequently filtered through a 0.45 μm syringe filter before testing. Polystyrene was used as the standard solution for calibration in a broad range of 0.5–1000.0 kDa. 2.4.3. Differential scanning calorimetry (DSC) To measure glass transition temperature and melting temperature of the composites, DSC was carried out with a TA DSC instrument (DSC Q2000, TA, America). The samples before and after degradation were heated from 0 °C to 210 °C at a speed of 10 °C/min. Afterwards, the samples were quenched and a reheating scan was carried out from 0 °C to 210 °C at a speed of 10 °C/min to get rid of thermal history and to determine the thermal transition of the scaffolds. The crystallinity degree (Xc) of the scaffolds was calculated according to the following Eq. (1) [2]:
Xc ¼ 2.2.2. Scaffold preparation Scaffolds were prepared by freeze-drying method [8]. PLLA was dissolved in 1,4-dioxane to obtain a 5% m/v PLLA/1, 4-dioxane solution. The fine aragonite powders were added to the PLLA solution resulting in a final polymer/powder weight ratio of 80/20. The solution was stirred for 15 min and sonicated for 30 min. Then the solution was transferred into a cylindrical mold and frozen at − 20 °C for 24 h. The cylindrical molds were lyophilized at −60 °C for 48 h. The PLLA/vaterite scaffold was fabricated the same way. The PLLA scaffold was prepared the same way without powder. Before tests, all scaffolds were dried in a vacuum oven for 3 days to get rid of the residual 1,4-dioxane.
ΔH m 100% ΔH ref
ð1Þ
where, ΔHm is the melting enthalpy for crystals of PLLA in the scaffolds, ΔHref is the melting enthalpy for pure crystals of PLLA. ΔHref = 90.95 J/g. 2.4.4. Water absorption and weight loss At each point of time, three samples of each scaffold were taken out of the PBS, wiped off the surface solution with filter paper and weighed to determine the wet weight (Ww). These samples were washed in ultra-pure water (Milli-Q), dried in a vacuum oven for 3 days and weighed to obtain the constant weight (Wd). Water absorption and
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weight loss of the scaffolds were calculated according to the following equations: Water absorption ¼
Weight loss ¼
W w −W d 100% Wd
W i −W d 100% Wi
ð2Þ
ð3Þ
where Wi is the original dry weight of the scaffold. 2.4.5. The mechanical properties The compressive strength and modulus of the composite scaffolds before and after PBS immersion were tested using a Zwick Z005 universal testing machine at ambient temperature. The samples were compressed at a rate of 1 mm/min until the strain was N 30%. The compressive strength was calculated by the maximum value of the linear part of the stress–strain curve [8]. The compressive modulus was determined by the slope of the initial linear region of the curve. Three samples for each scaffold were tested. By averaging the intermediate data the final result was determined. 2.4.6. SEM The bulk samples before and after degradation were dried in vacuum and fractured. The fractured morphology of scaffolds was observed using a scanning electron microscopy (SEM, FEI Quanta 200) at 30 KV after sputter coating with gold. 2.4.7. Mercury intrusion porosimetry The density and porosity of the scaffolds were assessed by a mercury intrusion analyzer (Autopore IV 9510, Micromeritics, America). All samples were weighed and kept in a cell under vacuum condition. At such lower pressure the pores of the samples cannot be intruded by mercury. Increasing pressures raised the amount of mercury entering the pores of the samples. The pressure of mercury was inversely proportional to the pore volume of the materials. The density was subsequently calculated by the weight and volume of the samples. The porosity was calculated according to the following Eq. (4) [30]: P l −P g ¼
4σ cosθ d
ð4Þ
where Pl is the pressure of mercury (Pa), Pg is the pressure of the gas (Pa), σ is the surface tension of mercury, θ is the contact angle and d is the pore diameter.
Fig. 1. pH of PBS containing the scaffolds or pearl powders. * shows significant differences between groups (p b 0.05). ** indicates significant differences between groups (p b 0.01).
Because vaterite is more easily dissolved than aragonite, PLLA/vaterite scaffold releases more alkalis into the PBS than PLLA/aragonite scaffold. The dissolution of CaCO3 from vaterite and aragonite produces an alkalinity in PBS, which could neutralize acid residues from PLLA and slow down the hydrolysis rate of PLLA [31]. 3.2. Concentration of Ca2+ in PBS solutions containing scaffolds or pearl powders Fig. 2 shows changes of the Ca2+ concentration in PBS containing the scaffolds or pearl powders. PBS and PLLA scaffolds do not release Ca2+. The concentration of Ca2 + in PBS containing vaterite powders is 0.13 mM on day 1 and increases slowly for 42 days, while in PBS containing aragonite powders, the initial Ca2+ concentration is 0.06 mM on day 1 and gradually rises to 0.20 mM within 28 days. As vaterite is more easily dissolved than aragonite, vaterite could release more Ca2+ and rapidly reach the high Ca2 + concentration on the first day. It is shown that the Ca2+ concentration is different for these two powders.
3. Results and discussion 3.1. pH of PBS containing scaffolds or pearl powders To investigate the degradation of the scaffolds and the pearl powders, pH changes of the solutions are measured (Fig. 1). The pH of PLLA stays at 7.40 in the first 20 days. By the 21st day a small observable decrease in pH occurs. The pH of PLLA remains constant below 7.40 during the degradation time. PLLA starts to degrade in PBS causing a change of pH. In contrast, pH of vaterite and aragonite powders rapidly increases to 7.80 and 7.65 on the 10th day respectively. The maximum pH value of vaterite powder is 8.60, remaining high all through the degradation time. Compared to pure PLLA scaffold and pearl powders, pH of PBS with composite scaffolds shows a different trend. In both composite scaffolds, the pH is higher than 7.40 and remained around 7.80 during the first 100 days. The dissolution of CaCO3 in composite scaffolds causes the gradual increase of pH. The degradation solutions containing PLLA/vaterite scaffold show a similar change of the pH value like PLLA/aragonite scaffold, but the pH of PLLA/vaterite scaffold keeps consistently higher than the pH of the PLLA/aragonite scaffold.
Fig. 2. Concentration of Ca2+ in PBS containing the scaffolds or pearl powders.
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Table 1 Molecular weight and molecular weight distribution of PLLA before and after degradation. Degradation time (days)
Scaffolds
Mw (Da)
0 100 100 100 200 200 200
PLLA PLLA PLLA/vaterite PLLA/aragonite PLLA PLLA/vaterite PLLA/aragonite
17.9 15.5 15.6 16.6 14.6 15.6 14.8
× × × × × × ×
104 104 104 104 104 104 104
Mn (Da) 12.7 8.1 9.0 9.5 2.8 2.5 3.4
× × × × × × ×
104 104 104 104 104 104 104
The two kinds of pearl powder show a similar size distribution from 0–23 μm (data not shown). Pearl powder easily aggregates to form small or large conglomerates in PBS, possibly affecting the dissolution of CaCO3. Composite scaffolds have a greater increase in releasing Ca2 +on day 7 and increase slowly after 7 days. Powders are more homogeneously dispersed in scaffolds than in PBS, therefore, composite scaffolds release more Ca2+ than pearl powders all through the degradation time. PLLA/ aragonite scaffold shows a similar change of the Ca2+ concentrations as PLLA/vaterite scaffold. Because vaterite is more easily dissolved than aragonite, Ca2+ concentrations of PLLA/vaterite scaffold keep consistently higher than of PLLA/aragonite scaffold. Buffer solutions are used to keep pH. PBS solution containing HPO2− 4 2+ and H2PO− concentration at a nearly con4 has abilities to stabilize Ca stant value. Previous works have studied the solubility of calcite and aragonite in seawater at 35‰ salinities, 25 °C and one atmosphere [32]. Calcite, aragonite and vaterite are three different CaCO3 crystals. Ksp values of calcite and aragonite are 10−6.37and 10−6.19 respectively. Ca2 + concentrations for calcite and aragonite could reach 0.653 mM and 0.804 mM. Because vaterite is easier to dissolve than aragonite, it is reasonable for higher Ca2+ concentrations of vaterite than aragonite. Thus, Ca2 + concentrations for composite scaffolds do not reach the saturation level for 42 days and just increase slowly by a buffering effect.
Mw/Mn
Peak of molecular weight (Da)
1.407 1.910 1.734 1.736 5.088 6.275 4.273
17.4 17.2 17.2 17.2 16.4 16.6 16.4
× × × × × × ×
104 104 104 104 104 104 104
Other peaks of molecular weight (Da)
1.1 × 104 1.0 × 104 1.1 × 104
scaffold corresponding to 1.0 × 104 or 1.1 × 104 Da. Meaning that some water-soluble PLLA oligomers may be separated from the scaffolds. PLLA in composite scaffold shows a relatively slower decrease of molecular weight compared with the pure PLLA scaffold. After 200 days molecular weight of PLLA in PLLA scaffold reduced from 17.9 × 104 Da to 14.6 × 104 Da, while in composite scaffold, it reduced to 15.6 × 104 in PLLA/vaterite and to 14.8 × 104 in PLLA/aragonite. It can be seen that compared to the PLLA scaffold, PLLA in composite degraded at a slower rate. The result is consistent with the pH change, meaning that alkalinity products from powders could provide a relatively steady environment decreasing the speed of PLLA degradation. The addition of powders into PLLA could decrease PLLA degradation rate. PLLA/vaterite scaffold shows a higher molecular weight of PLLA compared to PLLA/ aragonite scaffold. This may be highly related to the fact that more alkalinity products are released from vaterite powders than from aragonite
3.3. Molecular weight Changes of the molecular weight of PLLA in scaffolds during the degradation process are shown in Table 1. In general, all molecular weights of PLLA in scaffolds decrease during the degradation process. The molecular weight distribution (Mw/Mn) of PLLA in all scaffolds increases, which means a decrease in high molecular weight, with a subsequent increase in low molecular weight of PLLA. The peak of molecular weight of PLLA moved towards the lower molecular weight. PLLA in pure PLLA scaffold shows a dramatic decrease of molecular weight and a rapid increase of the molecular weight distribution (from 1.407 to 5.088). The chains fractured from its backbone and hydrolysis resulted in a decrease in molecular weight of PLLA. It often appears in the amorphous phase of PLLA [33]. It was reported that PLLA oligomers with a molecular weight below 104 Da are partially hydrosoluble [34]. On the 200th day there is another peak corresponding to 1.1 × 104 Da in the PLLA scaffolds. In composite scaffold, the low molecular weight PLLA also separated from PLLA Table 2 Characteristic thermal transition temperature of the scaffold before and after degradation according to the reheating scan. Scaffolds
Tg (°C)
Tm (°C)
ΔHm (J/g)
Crystallinity
PLLA PLLA/vaterite PLLA/aragonite PLLA 200 days PLLA/vaterite 200 days PLLA/aragonite 200 days
58.46 58.26 59.19 59.56 60.84 60.50
173.73 181.08 178.32 173.51 174.96 174.17
35.93 32.88 33.27 47.80 34.21 35.02
39.50% 36.15% 36.58% 52.55% 37.60% 38.50%
Fig. 3. DSC curves of the scaffolds before (a) and after 200 days degradation (b).
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Fig. 4. Water absorption of the scaffolds against degradation time.
powders. Thus, it could provide a steadier environment for declining PLLA degradation. 3.4. Differential scanning calorimetry (DSC) Table 2 shows that PLLA in PLLA scaffold has a glass transition temperature (Tg) of 58.46 °C and a melting temperature (Tm) of 173.73 °C, slightly differing from what can be found in literature [14]. After 200 days of degradation, Tg increased to 59.56 °C and Tm decreased to 173.51 °C. Fig. 3 also shows that the shape of the melting peak remains similar before and after degradation for PLLA scaffold, which indicates that there is no significant change in PLLA crystals after 200 days of degradation. With the addition of pearl powder into PLLA Tg decreases and Tm increases compared to PLLA. After 200 days of degradation Tg increases and Tm decreases in both composite scaffolds. Also, the shapes of melting peaks are changed. These results show that in composite scaffolds PLLA polymer has lower mobility after degradation and the PLLA crystals undergo some changes during degradation. The partially degraded main chains of PLLA may be the main factor for those characteristic temperature changes [14]. For two composite scaffolds, there are differences between Tg, Tm and the shape of the melting peak, which
Fig. 6. Change of the compressive strength (a) and compressive modulus (b) of the scaffolds before and after degradation. ** indicates significant differences between groups (p b 0.01). * shows significant differences between groups (p b 0.05).
may arise from different molecular weight, crystallinity, etc. Table 2 also shows the crystallinity degree of the scaffolds. For PLLA scaffold, there is an increase of crystallinity from 39.50% to 52.55% during the 200 days of degradation. PLLA crystallinity can be increased by the major way [2]: the amorphous PLLA would be the first disappearing in solution in the process of hydrolysis. Composite scaffolds display lower crystallinities compared to PLLA after the 200 days of degradation. The autocatalytic degradation of PLLA usually combines with acidic products, which could be buffered by the release of alkalinity products from powders [35]. As the molecular weight of PLLA in composite scaffold is higher than in PLLA scaffold (Table 1), alkalinity products from powders could provide a relatively steady environment decreasing hydrolysis of the amorphous phase of PLLA. 3.5. Water absorption
Fig. 5. Weight loss of the scaffolds against degradation time. ** indicates significant differences between groups (p b 0.01).
Fig. 4 shows the water absorption of the scaffolds. All scaffolds show similar water absorption during the incubation. In the first 3 weeks, water absorption of the scaffolds quickly increases with a maximum of about 900%. After 3 weeks, the water absorption of the scaffolds stabilizes at the saturation points. Although powders and PLLA are hydrophobic materials, the water absorption of scaffold gradually increases. Literatures have reported that a decrease in sample mass
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a
b
c
d
e
f
g
h
i
j
k
l
PLLA scaffold
PLLA/vaterite scaffold
PLLA/aragonite scaffold
Fig. 7. SEM images of fractured morphology of the scaffolds before and after degradation. (a, d, g, j) PLLA scaffold, (b, e, h, k) PLLA/vaterite scaffold, (c, f, i, l) PLLA/aragonite scaffold. The scale bar is 100 μm.
and an increase in sample dimensions will cause a rapid increase in water absorption [36]. The high porosity (N88%) and large pore size (about 100 μm) allow water to enter into scaffold more easily (data not shown). For PLLA scaffold, during the first period of degradation, PLLA absorbing PBS to some extent, leading in swelling and hydrolytic degradation of low molecular weight PLLA, and the increased defects and porosity will gradually increase water absorption. The main water-binding ability of composite scaffold is corresponding to PLLA swelling and the dissolution of pearl powder, which provide more room for water. This rapid increase of water absorption also causes the high weight loss of the scaffold and the possible porous structure destruction [31].
3.6. Weight loss Fig. 5 shows the weight loss of the scaffolds in PBS. For PLLA scaffold, with increasing degradation time, the weight loss is not changing linearly and is showing four steps. There is no weight loss of pure PLLA scaffold in the initial stage. After 2–4 weeks, a rapid increase of weight loss to 3% can be observed. Afterwards it tends to a steady condition during week 4 to 14. After 14 weeks another rapid weight loss occurs. PLLA is a semicrystalline polymer. Side chains fractured from PLLA backbone could reduce the PLLA weight during 2–4 weeks. However, the backbone keeps steady during week 4 to 14 and starts to fracture again after 14 weeks. At the same time, the hydrolysis of low molecular
weight PLLA from PLLA porous scaffold may be attributed to the change of the weight loss. The weight loss of the composite scaffolds is relatively faster compared to the PLLA scaffold. During 1 to 4 weeks the composite scaffolds show a higher weight loss, slowly increasing after 4 weeks. Considering the fast decrease in the mechanical properties of composite scaffolds (Fig. 6) and gradual increase of pH in PBS (Fig. 1), the dissolution of CaCO3 from pearl powders is the main factor for the weight loss of the composite scaffolds in the first 4 weeks. After 4 weeks there is a complex process of degradation because at the same time PLLA possibly degrades and pearl powder dissolves. Alkalinity products released from the dissolution of CaCO3 could decrease the process of hydrolysis and slow down the PLLA degradation rate [29]. Because vaterite is more easily dissolved than aragonite, it is reasonable for a higher weight loss of PLLA/vaterite than PLLA/aragonite in the first 3 weeks. After 3 weeks both composite scaffolds show similar weight loss while PLLA starts to degrade. 3.7. The mechanical properties The mechanical properties of the scaffolds were measured by compressive measurements. The compressive strength and compressive modulus of the three scaffolds show a different decreasing trend (Fig. 6). For the PLLA scaffold, the compressive strength and compressive modulus significantly decreased during the first 30 days but much slower afterwards. In the first 30 days, the hydrolysis of PLLA of
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structure. In conclusion, to destroy the structure of the PLLA scaffolds and composite scaffolds, 200 days is not long enough. 3.9. Porosity and bulk density As expected, bulk density decreases and porosity increases with proceeding degradation time (Fig. 8). Before degradation, the addition of pearl powder into PLLA increased the density of the scaffold (Fig. 8(a)). Such high density can improve the mechanical properties of the scaffolds [37]. PLLA scaffold shows a dramatic decrease in density after the degradation. The hydrolysis of low molecular weight PLLA from PLLA porous scaffold, which may be an amorphous phase, contributes to the decrease of density. Because the mechanical properties of PLLA scaffold are mainly derived from both its porous structure and density, the observed change of density does not match the changes of the mechanical properties of the scaffolds after 30 days. In contrary, the composite scaffold shows a continuous decrease in density at a relatively slow speed, which is mainly caused by the dissolution of the pearl powder. For PLLA/vaterite scaffold the porosity increased from 88% to 97% after 200 days degradation (Fig. 8(b)). In contrast, for PLLA/aragonite and PLLA scaffold porosity reaches a maximum after 100 days and is decreasing afterwards again. The dissolution of the powder and the hydrolysis of PLLA increased the porosity. However, the relation of the appearance and disappearance of pores in the porous scaffold should be considered. The dissolution of pearl powder will increase porosity, while collapsing pores decrease porosity. Alkalinity products released from PLLA/vaterite scaffold could drop the PLLA degradation rate even more. The decrease of the amount of hydrolysis of PLLA will decline the number of collapsed pores. At the same time, since vaterite is more unstable and more easily dissolved than aragonite and PLLA, the dissolution of vaterite powders in scaffolds could cause a steady increase of porosity for up to 200 days. For the PLLA/aragonite scaffold, the balance between the dissolution of aragonite powder and collapsed pores may determine the final porosity of the scaffold. 4. Conclusions Fig. 8. Bulk density (a) and porosity (b) of different scaffolds during degradation. * shows significant differences between groups (p b 0.05).
low molecular weight from PLLA porous scaffold contributes to the decrease of compressive strength and compressive modulus of PLLA scaffold. After 30 days, the slight decrease of compressive strength and compressive modulus is derived from a steady porous structure of PLLA. The composite scaffolds display a gradual decrease of the compressive strength and compressive modulus. After 200 days, the compressive strength is still higher than that of the PLLA scaffold before degradation. The compressive modulus is slightly less than that of the PLLA scaffold before degradation. During the 200 days of degradation, the dissolution of pearl powders may be the main factor for a decreasing compressive strength and compressive modulus. At the same time, the dissolution of pearl powders and the hydrolysis of PLLA increasing defects will result in losing the order of whole porous structure and decreasing the mechanical properties of the scaffolds. 3.8. SEM Fig. 7 shows that there are some little holes appearing on the surface of the pore walls after degradation. With ongoing time, surface roughness of the pore walls increased in all scaffolds. For the PLLA scaffolds, the hydrolysis of PLLA and the recrystallization of partially degraded short chains of PLLA may have increased the surface roughness [2]. For composite scaffolds, the partial dissolution of pearl powders, the degradation of PLLA, and recrystallization onto the surface cause this roughness and irregular morphology. All samples maintain their porous
In this work, different degradation behaviors of PLLA, PLLA/vaterite and PLLA/aragonite scaffolds in PBS have been studied. The gradual increase of pH in composite scaffolds can decrease the hydrolysis rate of PLLA. Moreover, composite scaffolds show a greater ability to release Ca 2 + than pearl powders in PBS. During the degradation, PLLA cooperating with pearl powders displays changes such as mechanical properties, weight loss, bulk density and porosity. Alkalinity products released from composite scaffolds can provide a relatively steady environment for slowing down the degradation rate of PLLA and prolonging the degradation time of composite scaffolds. PLLA/vaterite and PLLA/ aragonite scaffolds show similar degradation behaviors. Further investigation could be performed on biological properties including degradation behavior in vivo and biocompatibility in order to promote the novel material in clinical applications. Acknowledgments The authors are grateful for the financial support from the National Natural Science Foundation of China (51361130032) and the Doctor Subject Foundation of the Ministry of Education of China (20120002130002). References [1] K. Madhavan Nampoothiri, N. Rajendran Nair, R. Pappy John, An overview of the recent developments in polylactide (PLA) research, Bioresour. Technol. 101 (2010) 8493–8501.
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