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The Effects of Bioactive Nanoparticles on theDegradation of DLGAEsperanza Díaz a , Igor Puerto a & Iban Sandonis aa Departamento de Ingeniería Minera , Metalúrgica y Ciencia de Materiales, Universidad delPaís Vasco (UPV/EHU) , Portugalete , SpainPublished online: 11 Sep 2014.

To cite this article: Esperanza Díaz , Igor Puerto & Iban Sandonis (2015) The Effects of Bioactive Nanoparticles onthe Degradation of DLGA, International Journal of Polymeric Materials and Polymeric Biomaterials, 64:1, 38-46, DOI:10.1080/00914037.2014.886242

To link to this article: http://dx.doi.org/10.1080/00914037.2014.886242

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The Effects of Bioactive Nanoparticles on the Degradationof DLGA

ESPERANZA DIAZ, IGOR PUERTO, and IBAN SANDONIS

Departamento de Ingenierıa Minera, Metalurgica y Ciencia de Materiales, Universidad del Paıs Vasco (UPV=EHU),Portugalete, Spain

Received 30 October 2013, Accepted 19 January 2014

The effect of bioactive nanoparticles on the in vitro degradation of DLGA composite scaffolds is investigated. Fabricated bythermally induced phase separation, the scaffolds present a high porosity (>90%). In vitro degradation is performed by immersingthe scaffolds in a phosphate buffered saline solution, to evaluate water absorption, pH change, and weight loss. Chain scission byhydrolysis reduces the average molecular weight and increases the polydispersity index. The incorporation of modified hydroxya-patite nanoparticles significantly affects the DLGA degradation process, inducing appreciable changes in the morphology of thematerial, but not in its percentage of porosity. Nanohydroxyapatite blocks the entry of water, reducing the degradation rates.

Keywords: Biodegradable polymer, degradation, in vitro, nanohydroxyapatite, pH, scaffolds

1. Introduction

Organ and tissue loss or failure resulting from an injury orother type of damage is a major human health problem.

Tissue engineering is an interdisciplinary research field thatincludes all processes involved in the development of biologi-cal substitutes that restore, maintain, or improve tissue func-tion. Basic tissue engineering requires a suitable cell sourceand an appropriate material structure (scaffold) onto whichthe cells may be seeded and grown [1,2]. Scaffolds arethree-dimensional porous biomaterials designed to providean initial mechanical support and a three-dimensional nichefor transplanted cells [3], which promote cell adhesion and

Address correspondence to: Esperanza Dıaz, Departamento deIngenierıa Minera, Metalurgica y Ciencia de Materiales, Uni-versidad del Paıs Vasco (UPV=EHU), Marıa Dıaz de Haro,68, 48920 Portugalete, Spain. E-mail: esperanza.diaz@ehu.es

International Journal of Polymeric Materials and Polymeric Biomaterials, 64: 38–46

Copyright # 2015 Taylor & Francis Group, LLC

ISSN: 0091-4037 print/1563-535X online

DOI: 10.1080/00914037.2014.886242

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extracellular matrix (ECM) deposition and permit sufficienttransport of gases, nutrients, and regulatory factors to allowfor cell survival, proliferation, and differentiation. More-over, they are also expected to be mechanically strong, mal-leable, bioactive, and biodegradable [4–7].

The material used to fabricate these scaffolds must fulfill aseries of physical and chemical requirements. Biodegradabilityis very critical factor and should be verified at a controllablerate that approximates the rate of tissue regeneration andprovokes a minimal degree of inflammation or toxicity.

Polymers can offer unique properties such as a high surfaceto volume ratio, high porosity with very small pore size,controllable biodegradability, and goodmechanical behavior.Moreover, they offer better chemical versatility than anyother material, to match the physical and mechanicalproperties of various tissues or organs of the body [8].

There are two kinds of polymer materials, which may bederived from either natural sources or from synthetic organicprocesses. Synthetic polymers have the advantage of beingproduced in large uniform quantities and are normallycheaper and have a longer shelf life than natural polymers.Aliphatic polyesters, such as poly(L-lactic acid) and its copo-lymers with D-lactic acid and glycolic acid are widely used forscaffold fabrication.

Poly(lactide-co-glycolide) (DLGA) is a very popularbiodegradable polymer, which has the approval of the U.S.Food and Drug Administration for human clinical applica-tions and combines good mechanical properties, toughness,excellent processability, and adjustable degradation rate.

DLGA can be obtained via ring-opening copolymeriza-tion of lactide and glycolide. Copolymers can be fabricatedusing both L and D,L-lactide. D,L lactide copolymers areamorphous and offer good mechanical properties. Thedegradation kinetics of DLGA are influenced by its relativemolecular mass, comonomer ratio, specimen size, configur-ation, and environmental conditions, among other aspects;the intermediate copolymer being much more unstable thanthe homopolymers. DLGA has been widely used in the fieldof control drug release [8,9]. It is a biodegradable polymerand follows a bulk degradation mechanism when immersedin a neutral aqueous solution such as PBS solution. DLGAdegrades via simple hydrolysis of ester bonds into itsmonomeric form, producing lactic and glycolic acids, whichare eliminated from the body by incorporation into the tri-carboxylic acid cycle by normal metabolic processes. Thedegradation is crucial to scaffolds in tissue regeneration.The rate of degradation can affect some cellular processessuch as cell growth, tissue regeneration, host response, andmechanical properties.

One of the limitations that have been found regardingthe use of these polymers in tissue engineering applicationsis the decrease in pH that is observed in the surroundingsof the implant, which is produced by the release of acidicdegradation products. Several studies have reported adverseeffects caused by these processes such as inflammatoryresponses [10].

A way to reduce the acidity of the degradation products ofthese kinds of polymers could be by neutralizing them with analkaline substance. In this approach, a bioactive ceramic such

as HA (hydroxyapatite) may be of interest. Hydroxyapatite(HA) is a major component in bones and is widely used as abioactive ceramic, because it shows good osteoconductivityand forms strong chemical bonds with bone. It has an alkalinepH and shows poor mechanical properties, which counts asone of its disadvantages. HA has been successfully blendedwith poly lactic polymers to form composite biomaterials,for the fabrication of porous scaffolds for bone replacementpurposes. Recent research has found that the biocompatibil-ity of these composite scaffolds is better, in addition the useof nano sized HA may have other special properties due toits small size and huge specific surface area [11,12].

In this paper, thermal induced phase separation (lyophili-zation) was employed to prepare DLGA scaffolds andDLGA=nHA composite scaffolds. The objective of thisinvestigation is to study in vitro degradation of DLGA scaf-folds and DLGA=nHA composite scaffolds, which may notonly lead to bioactive scaffolds, but may also present apotential to offset the decrease in pH in phosphate buffersolution (PBS). The nanohydroxyapatite particles act as aphysical barrier and block the entry of water causing adecrease in the scaffold degradation rates.

2. Experimental

2.1 Raw Materials

Poly DL-lactide and glycolide (DLGA) copolymers in a47=53 molar ratio were supplied by PURAC (PURASORBPDLG5004, the Netherlands) and purified by dissolution inchloroform. The weight-average relative molecular weightMw¼ 94800, Mn¼ 65600, and polydispersity Mw=Mn¼1.4452 of DLGA were determined using gel permeationchromatography (GPC; Perkin Elmer 200) in THF. GPCwas performed with a tetrahydrofuran solvent using a reflec-tive index detector of Perkin Elmer 200 as the detector.Calibration was done in accordancewith polystyrene standardswith a flow rate of 1mL=min. Nano hydroxyapatite (nHA) wassupplied by Aldrich Chemistry (USA), with a particle size>200nm and Mw¼ 502.31g ml�1. 1,4 Dioxane purchasedfrom Panreac p.a. (Barcelona, Spain) was used as solvent.PBS in water, supplied by Fluka Analytical (Sigma Aldrich,USA) at a pH of 7.2, was used as the degradation fluid.

2.2 Fabrication of Porous Scaffolds

Pure DLGA and DLGA=nHA composite scaffolds werefabricated by TIPS (thermally induced phase separation)followed by a freeze-drying technique. Briefly, DLGA wasdissolved in 1,4 dioxane in a proportion of 2.5% (w=v), bystirring for 2 h at a temperature of 50�C. After its completedissolution, the resultant solution was poured into alumi-num moulds. At this step, nHA was blended by ultrasonicstirring for 5min, in proportions of 10%, 30% and 50% oftotal polymer mass, to form the composite scaffolds. Thesolutions were frozen and freeze-dried for several days toextract the solvent completely. Foams such as porous scaf-folds with porosity of up to 90% were obtained by thismethod.

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2.3 In Vitro Degradation

Samples for degradation were cut into 0.5 cm2 rectangularpieces and weighed. After that the specimens were placedin identical glass test tubes containing 10ml of PBS, totallyimmersed and incubated in a thermostated oven at 37�C andunder static conditions. After selected degradation times (1,2, 4, 6, and 8 weeks), the specimens were recovered, carefullywiped to remove surface water, and weighed to determinewater absorption. The pH change in degradation mediumwas determined using a pH meter PCE 228 by PCE Instru-ments (Spain) and corrected by temperature. Finally, thesamples were dried over two weeks to a constant weight thatwas recorded in order to determine the weight loss.

2.4 Characterization

2.4.1 Water Absorption and Weight Loss

Water absorption and weight loss were evaluated by weigh-ing. The percentage of water absorption Wa% was calculatedby the following equation:

Wa% ¼ Ww �Wr

Wr� 100 ð1Þ

where Ww is the weight of the wet=swallow specimen afterremoving surface water and Wr is the residual weight of acompletely dry sample after degradation. Weight loss per-centage (WL%) was estimated with the following equation:

WL% ¼ W0 �Wr

W0� 100 ð2Þ

the original mass of the sample was designated as W0.

2.4.2 Mercury Pycnometry

The porosity of the scaffolds was quantified by mercury pyc-nometry. To do so, the scaffolds were dipped one by one in acontainer of mercury, placed on electronic scales with thehelp of a metal device. Knowing the density of mercury(qHG¼ 13.57 g cm�3) and the mass indicated by the scale,we may calculate the volume of the mercury (VolHg). Thevolume displaced by the mercury is equivalent to the volumeof the sample in question. So, knowing the initial mass andVolHg thereof (Msa), the bulk density (qa) may be calculatedwith the following equation:

qa ¼ Msa=VolHg ð3ÞUsing both bulk density and the density of the polymer

(qp), measured by pycnometry on the pulverized material,the percentage porosity was calculated by the followingequation:

%P ¼ ð1� qa=qpÞ � 100 ð4Þ

where P is the percentage porosity. Measurements weremade for each material.

2.4.3 SEM Analysis

The bulk morphology of the scaffolds was examined usingscanning electron microscopy (SEM; HITACHI S-3400N,Tokyo, Japan). Prior to analysis, the samples were coatedwith a layer of gold, in a JEL Ion Sputter JFC-1100 at

1200V and 5mA, to avoid sample charging under theelectron beam.

2.4.4 DSC Analysis

The thermal characteristics of the polymer were determinedusing differential scanning calorimeter (DSC TA Instru-ments) equipped with an intracooler. Approximately 10mgof polymer was placed in a crimp-sealed DSC hermeticaluminum pan. A nitrogen purge gas was used to preventoxidation of the samples during the experiments, which weresubjected to temperature scans ranging between �20�C and200�C at temperature=time ratios of 10�C=min.

3. Results and Discussion

3.1 Molar Masses and Polydispersity Changes

The temporal variation in the Mw, Mn, and the polydisper-sity of DLGA and DLGA=nHA composite are shown inFigure 1. The Mw of the DLGA decreased during the entiredegradation process. However, its rate of decrease differedin the case of each different scaffold. The molecular weightof the DLGA decreased more rapidly than those of theDLGA=nHA composites. The molecular weights (Mw)gradually decreased in both cases, indicating that the degra-dation occurred from the beginning of the period of immer-sion, although the composite scaffolds degraded very slowly.The fact that the composite material lost less molecularweight due to the presence of nanoparticles could be dueto a well-adjusted buffer effect at the DLGA=nHA interface,the homogeneity of which prevented the penetration of PBS[10]. This behavior differed greatly from that reported byother authors, which was probably due to the incorporationof nHA particles and the manufacturing process of the scaf-folds (lyophilization). The degradation is a bulk mechanism,catalyzed by carboxyl end groups that are formed by therupture of polymer chains, which can therefore impede theautocatalytic nHA process. In consequence, both the rateof degradation and the molecular weight loss values arelower (see Figure 2).

The polidispersity index of all DLGA and DLGA=nHAshowed changes during the degradation process. The valuesof the DLGA scaffolds changed from 1.44 to 2.07 and thoseof the DLGA=nHA composite scaffolds varied from 1.44 to3.30. Three peaks were observed in the chromatograms forthese composites, at approximately three weeks into thedegradation study, and the most prominent peak was selec-ted for measurement. The polydispersity increase within vitro degradation time was due to cleavage of the polymerchains as a result of hydrolysis [11–14].

3.2 Thermal Analysis

During heating, the polymer undergoes transition from aglassy to a rubbery state at the glass transition temperature(Tg). The poorly organized structure of the amorphousDLGA, required less heat to make this transition than thesemicrystalline or crystalline structures. In Figure 3a we cansee the results obtained by Differential Scanning Calorimetryfor a sample and for scaffolds DLGA,DLGA=nHA 10%, and

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DLGA=nHA composite scaffolds 50% without degrading.The DLGA used for this study is a Poly DL-lactide andglycolide (DLGA) amorphous copolymer in a 47=53 molarratio. Looking more closely at this figure shows us that theintroduction of nHA favors the rigidity of the polymer chains,which produces a considerable increase in the glassytransition temperature that rises from 18.2�C for samples ofscaffolds to about 30�C for the composite [14,15].

In an in vitro degradation process, which we studied, areduction of the molecular weight by chain cleavage in thehydrolysis process might be expected to occur. A given massof polymer would imply higher fractions of chain-ends andhence increased free volume (i.e., the reduction in overallflexibility should increase the value of Tg). However, thestiffness caused by high amounts of nHA incorporated inthe composites counteracted this effect and caused the Tg

Fig. 1. (a) Changes in Mw for DLGA and DLGA=nHA 30%, as a function of degradation time. (b) Changes in Mn for DLGA andDLGA=nHA 30%, as a function of degradation time. (c) Changes in polydispersity index, for DLGA and DLGA=nHA 30%, as afunction of degradation time.

Fig. 2. Rupture of DLGA chains in the degradation.

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to go from 30�C for samples with a higher content of nHAto 40�C for the same samples with a degradation time of6–8 weeks [15] as seen in Figure 3b.

3.3 FTIR

The nHA were initially analyzed by IR. The characteristicbands of c2 PO3�

4

� �were observed at 566 and 601 cm�1, c1

PO3�4

� �at 954 cm�1, and c3 at 1087 and 1022 cm�1. These

reflections indicate the classification of the polyhedrons of

PO3�4 in the glass structure (see Figure 4) [12,16]. Looking

at the spectra of the DLGA DLGA=nHA, we can see thatthere is apparently no absorption band that has altered theirposition or intensity, so that we can say that the polymer doesnot interact with nanohydroxyapatite DLGA homogeneouslybut is dispersed in the dough, which will be confirmed in theSEM observation. This is contrary to the results obtainedby other authors with PLLA=nHA composite scaffolds usingthe manufacturing process of electrospinning [12]. In samplesof scaffolds and DLGA scaffolds and DLGA=nHA com-posite scaffolds at different weeks of degradation, we can

Fig. 4. (a) FTIR spectra of nHA, DLGA, and DLGA=nHA.(b) FTIR spectra of DLGA=nHA 10% after various degradationtimes.

Fig. 3. (a) DSC thermograms of DLGA, DLGA=nHA 10%, andDLGA=nHA 50% after various degradation times. (b) DSCthermograms of DLGA=nHA 50% after various degradationtimes.

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observe the appearance of a new band (very broad for thecontribution of water absorption) at around 3350 cm�1 thatcan be attributed to alcohol groups formed during the exci-sion of the polymer chains by the hydrolysis of DLGA intolactic acid and glycolic acid. Moreover, the peak correspond-ing to 1750 cm�1 (C=O stretching) dims when degradationbreaks up the chains over longer periods of time.

When the degradation process starts both for DLGA andfor DLGA=nHA composite scaffolds, an absorption bandappears that corresponds to COO� asymmetric stretching at1600 cm�1 that cannot be seen in the non-degraded scaffolds,which is due to the hydrolysis process of the polymer chains.

3.4 pH Variations

DLGA degradation is mainly achieved via chemical hydroly-sis and a low pH or a very high pH causes significant catalysisof the hydrolysis of an ester bond. The pH of the buffer sol-ution for the DLGA scaffolds continuously decreased andreached 6.78 after 6 weeks and the 6–8 weeks remained con-stant (see Figure 5). Any decrease in pH showed an increaseddegradation rate. In the case of the DLGA=nHAp compo-sites, the pH also continuously decreased to values close to6.91 for the sample with less nHAP (10%) and to 6.97 forsamples with more nHAP (50%). But, in all of cases, the ratesof degradation were less than the DLGA and the pH werehigher. In vitro investigations have shown that the microen-vironment within the acid created during DLGA degradati-on, which is degraded by simple hydrolysis of ester bonds,lactic acid and glycolic acid, in an accelerated processcatalyzed by the generation of carboxylic acid, results in adecrease in molecular weight [17,18].

The addition of salts such as hydroxyapatite or sodiumbicarbonate in the matrix caused neutralization of the acidreleased by DLGA. These particles are propitious to moder-ate inflammation from the acid released by autocatalyticalacceleration of DLGA, which could translate into anabsence of live adverse response in tissue [12].

Fig. 5. pH change of phosphate buffer solution against degra-dation time.

Fig. 6. Water absorption, for DLGA and DLGA=nHA com-posite scaffolds, as a function of degradation time.

Fig. 7. Weight loss of DLGA and DLGA=nHA composite scaf-folds against degradation time.

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3.5 Water Absorption

Figure 6 shows the percentage of absorbed water versus thedegradation time (in weeks). The percentage of absorbedwater increased over the degradation time in all samples.DLGA is a pure sample, which absorbs more water. Bycomparing these results with those of Figure 1, we see that

the more water the sample absorbs, the more rapidly it isdegraded (accelerated weight loss). The sample with thelarger content of nHA are unable to stabilize water consump-tion over the first three weeks of the degradation process.

The water absorption process is a balance betweenthe dissolution of oligomers in solution and the material

Fig. 8. SEMobservation of surface morphology of DLGA. (a) DLGA before degradation. (b) DLGA=nHA 10% before degradation.(c) DLGA after degradation in vitro for three weeks. (d) DLGA=nHA 10% after degradation in vitro for three weeks. (e) DLGA afterdegradation in vitro for one week. (f) DLGA=nHA 10% after degradation in vitro for six weeks.

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consumption PBS residue. An increase in water uptake reflectsthe degradation rate in the initial state [18]. The accumulationof hydrophilic degradation products inside the scaffold leadsto an increase in water absorption during the degradation pro-cess. When the absorption of water reaches a certain value, thespeed of absorption is reduced as a result of the dissolution ofdegradation products. These products introduce nHA parti-cles, which slow down the rate of degradation of the DLGAscaffolds, because they are an alkaline solution, which actsas a physical barrier that blocks the entry of water [12,19]and causes a decrease in the rate of degradation.

Porous scaffolds made from amorphous poly ester–likeDLGA are often regarded as hydrophobic biomaterials,the hydrophobicity of which blocks the absorption of waterand leads to degradation by cleavage of hydrolytically sensi-tive ester bonds. A higher content of less hydrophobic GAunits in the copolymers (as is our case) facilitates the absorp-tion and diffusion of water and thus the hydrolysis. Not onlycould the copolymer composition affect the degradationbehavior, but so could the additives in the scaffolds. Forexample, the incorporation of nHAp nanoparticles inDLGA scaffolds adjusted the acidic degradation of DLGA.

3.6 Percentage Weight Loss

From a close look at Figure 7, we can see how the percent-age weight loss increases almost exponentially until thefourth week of degradation for samples and DLGA andDLGA=nHA (10%), after which its increase is almost linear.This decrease in the degradation rate may correspond to alarge accumulation of degradation products and their dissol-ution. In general the value of weight loss for all the samplesunder study increased as degradation increased over time.However, unlike the conclusions of other authors [11,19]weight loss in the degradation process became smaller withincreasing concentrations of nHA particles.

We can see how this loss is stabilized in the sample withthe highest concentration of nHA at somewhere between 6and 8 weeks, reaching a weight loss of about 34% comparedto the weight loss of DLGA without nHA that was 78%.Some authors have proposed that degradation is faster andhigher with increasing water consumption for the poly lac-tides and bulk copolymer degradation mechanism [13,19].

The introduction of nHA particles slows the weight loss ofDLGA scaffolds because they act as a physical barrier thatblocks the entry of water [12] and causes a decrease in the rateof weight loss and consequently the degradation rate.

3.7 SEM

Morphological changes of the DLGA and DLGA=nHAcomposite scaffold were determined by SEM microscopyobservation. The addition may be seen in Figure 8 of parti-cles that reduce the size of the pores, but do not appear togreatly affect the high percentage of porosity (Figures 8aand 8b). The particles are uniformly distributed in the poly-mer matrix and are included before the lyophilization pro-cess in which these porous supports are made. The highlyporous scaffolds or those with a smaller pore size degrade

more slowly than those with larger pore sizes or with fewerpores and thicker walls, which decrease diffusion of acidicdegradation products and therefore improve acid hydrolysis.When comparing samples with DLGA and DLGA=nHA,we can see (Figures 8c and 8d) that the latter degrade moreslowly than the DLGA [11,18]. Micropores were observedon the walls of the DLGA scaffolds over the first week ofdegradation (see Figure 8e), which is probably a morpho-logical feature that increases the degradation rate. Overthe first week of degradation, the surface morphology canbe seen to change from a smooth to an abrupt surface. Thismay be due to degradation, because the porous surfaces ofthe degradation products are released and part of the nHAparticles are exposed outside the scaffold walls (seeFigure 8f), resulting in a rougher surface [18,20]. The forma-tion of these precipitates on the composite surface maystimulate and enhance cell-material interactions and there-fore their biological response, so that the filler material couldbe used where bone regeneration is needed.

4. Conclusions

The following conclusions may be drawn from the resultsthat have been presented. Three-dimensional DLGA andDLGA=nHA composite scaffolds with a porous structureand a porosity of over 90% were fabricated by thermallyinduced phase separation. nHA particles were uniformlydistributed in the polymer matrix and did not appear toaffect the percentage porosity although its morphologywas affected. The effect of bioactive nanoparticles on thein vitro degradation of these scaffolds translates into lessvariation in pH, and a decrease in the rate of degradationcan be observed in the values obtained for Mw, Mn, and %weight loss. The nanohydroxyapatite particles acted as aphysical barrier and blocked the entry of water, causing adecrease in the rate of degradation of the scaffolds.

Acknowledgments

Technical and human support provided by SGIker (UPV=EHU, MICINN, GV=EJ, ERDF, and ESF) is reallyappreciated.

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