Immobilizing hydroxyapatite microparticles on poly(lactic acid) nonwoven scaffolds using layer-by-layer deposition

Abstract

A composite porous scaffold for bone cell culture was fabricated by immobilizing HA (hydroxyapatite) (Ca₁₀ (PO₄)₆ (OH)₂) microparticles on PLA (poly(lactic acid)) nonwoven fibers using the layer-by-layer deposition technique. A nonwoven PLA of thickness 1 mm, with average pore size of 230 µm and a porosity of 94%, was used. The nonwoven was functionalized with aqueous 65% deacetylated chitosan followed by rinsing, and then a second padding with aqueous sodium alginate loaded with varying percentages of HA microparticles (0.01%, 0.1% and 0.2%), resulting in a composite porous nonwoven. Sodium alginate was revealed to be an efficient polymer for obtaining a stable dispersion of the HA microparticles in an aqueous medium.

Atomic force microscopy and scanning electron microscopy images, zeta potential and wettability tests showed successfully the different surface modifications occurring at each step of surface functionalization. The chitosan coating cationized the PLA fiber surface, providing good adhesion of the HA-loaded anionic alginate coating. HA was almost homogeneously distributed at the PLA fiber surfaces with only a small reduction in the scaffold porosity, which reached 75%.

The composite PLA/chitosan/alginate/HA nonwoven structures were tested as scaffold for adhesion and proliferation of rat pre-osteoblast MC3T3-E1 cells. The results showed that higher loading with HA improved the MC3T3 cell adhesion and proliferation after 3 and 6 days of culture.

Keywords

poly(lactic acid) fibers nonwoven alginate chitosan padding layer by layer hydroxyapatite osteoblast cell culture

For healing of some bone diseases (bone cancer and periodontal disorders), the use of porous scaffolds is necessary.¹ Since bone defects are irregular in shape and are of different sizes, bone scaffolds should have the capacity to be manufactured in varieties of forms and sizes. Lots of research works deal with electrospun nanofibers, especially poly-L-lactide (PLLA), poly (lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) nanofibers loaded with hydroxyapatite (HA),^2–8 which are ideal in surface roughness to promote initial cell adhesion,³ but have limited pore size. Recently, Murphy et al.,⁹ Ducheyne et al.¹⁰ and Lanza et al.¹¹ showed that cellular infiltration and nutriment diffusion provided by scaffolds with larger pore sizes from 100 to 600 µm are needed for bone tissue repair.

Indeed, conventional nonwovens based on microfibers having pore diameters in the range of 30–300 µm, have improved mechanical properties compared to electrospun nonwoven webs, and may provide an appropriate scaffold for bone cell culture. Fiber functionalization with HA (Ca₁₀(PO₄)₆(OH)₂)¹² is necessary to promote bone cell adhesion and proliferation.

During bulk functionalization of fibers with HA, dispersion of HA in the polymer matrix poses problems.¹³ Surface modifiers such as surfactants or silanes^14,15 used to improve dispersion of HA particles in the polymer matrix are not always nontoxic and biocompatible.¹⁴

Recently, some researchers have used alginate to disperse nano cockle powder.¹⁶ Thus, in our study, alginate polymer was chosen to disperse HA microparticles for surface functionalization of poly(lactic acid) (PLA) nonwoven fibers. Alginate is a natural polysaccharide found in seaweed, and unlike collagen does not present the risk of contamination by allo- or xeno-proteins or viruses. Alginates are linear copolymers of 1-4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G). The monomers are arranged in a blockwise pattern along the chain with homopolymeric regions of M and G, termed M- and G-blocks, respectively, interspaced with regions of alternating structure (MG-blocks) (see Figure 4).^17–20 This polymer has been shown to promote adhesion and proliferation of bone cells.^17–19

Thus, in our study, the potential use of sodium alginate polymer to immobilize HA particles on a PLA nonwoven fiber web with average pore dimension of 230 µm was investigated. A nonwoven structure of thickness 1 mm, based on PLA microfibers (average diameter 30 µm) was first processed. Since in a previous study, alginate (polyanionic) polymer has been shown not to adhere effectively on the surface of a hydrophobic negatively charged polyester (PET) fiber surface,²⁰ a layer-by-layer deposition technique with a prior deposition of chitosan followed by the deposition of alginate biopolymer loaded with HA was carried out. Indeed, chitosan polymer has already been shown to promote bone growth. In this study, the prior chitosan deposition was aimed at cationizing the PLA fiber surface for improved adhesion of the anionic alginate polymer.

Various percentages of HA (0.01%, 0.1% and 0.2%) were dispersed separately in aqueous alginate solution before deposition on chitosan-coated PLA fibers.

The functionalized nonwoven webs were characterized by physico-chemical measurements: zeta potential, water contact angle (WCA), capillary uptake, scanning electron microscopy (SEM), atomic force microscopy (AFM) and the air permeability test. Biological tests were carried by measuring the number of pre-osteoblast cells MC3T3/E1 in the scaffolds after 3 and 6 days of culture. The use of the composite fibrous nonwoven PLA was assessed for future applications in bone tissue engineering.

Materials and methods

Materials

Manufacturing of nonwovens with PLA microfibers

PLA (NatureWorks®) grade for fibers (M_n_(PLA) = 44,900 g/mol; M_w/M_n = 1.9; D-isomer ≈ 2% and L-isomer = 98%) was kindly supplied by NatureWorks LLC. Cylindrical shaped PLA fibers of diameter 30 µm were manufactured at the GEMTEX laboratory using the spinning device Spinboy I from Busschaert Engineering. PLLA pellets were molten at 215℃ and extruded through a die consisting of 40 channels with a diameter of 400 µm to obtain a multifilament yarn that was then coated with a spin finish (Crosanol I-PA07, Eurodye). The multifilament yarn was then hot drawn between two rolls to form textured filaments.

The nonwoven was then manufactured at the CENT (Centre Européen des NonTissés) nonwoven prototyping platform (IFTH), using a F.O.R. carding system (Italy) and a Perfojet spunlacing machine for fiber web consolidation. Carding is the mechanical action in which the fibers are held by one surface while the other surface combs the fibers (using wires), causing individual fiber separation. There is a process of parallelization followed by delivery of fibers in the form of a web. In spunlacing, the consolidation of the web is done by fine water jets under very high pressure, entangling the fibers in the depth direction. The nonwoven formed had an average thickness of 1 mm, an areal density of 139 g/m², a porosity of 94% and an average pore size of 230 µm.

Cleaning of nonwovens to remove the spin finish

A spin finish is added onto fibers to improve their processability during the spinning and carding processes, and in particular to avoid breakage of fibers. Without removal of this spin finish, no cell adhesion occurred owing to the cytotoxicity of the spin finish. The nonwoven was cleaned to be free from spin finish before surface functionalization and biological characterization. A standard method developed in our surface chemistry laboratory was used: the nonwoven sample was subjected to Soxhlet washing with petrol ether for 2 hours, dried at room temperature to evaporate all solvent, then washed again with ethanol for 24 hours using Soxhlet and then dried for 24 hours. The samples were then subjected to three successive rinsings using pure water at room temperature (duration of 15 minutes). The surface tension of the last rinse water was measured and was around 72.3 mN/m, which is the surface tension of pure water. This confirmed the removal of all spin finish impurities from the PLA fiber surface.

Chitosan polymer

A total of 65% deacetylated low molecular weight chitosan (purchased from Sigma Aldrich) was used. An aqueous solution of 3 g/l of chitosan was prepared using distilled water acidified to pH 5 using acetic acid.

Alginate polymer

Low molecular weight sodium alginate polymer (with a viscosity of 250 cps at 2% aqueous alginate) was purchased from Sigma Aldrich.

Hydroxyapatite

Calcium phosphate powders were prepared at LMCPA, Laboratoire des Matériaux Céramiques et Procédés Associés (Maubeuge-France) by the aqueous precipitation technique^21,22 using a diammonium phosphate solution (NH₄)₂HPO₄ (Carlo Erba, France) and a calcium nitrate solution Ca(NO₃)₂·4H₂O (Brenntag, France). For HA (Ca₁₀(PO₄)₆(OH)₂) powder synthesis, the temperature was set at 50℃ and pH 11.

The Ca/P ratio was determined by powder X-ray diffraction analysis (Rigaku Miniflex) using the intensity ratio of lines HA (211) I TCP (tricalcium phosphate) (0210) according to the method of the proportioned additions,²³ and was equal to 1.667 (Figure 1). The presence of calcium pyrophosphate, (Ca₂P₂O₇) was checked by infrared spectroscopy on a Fourier transform spectrometer (Jasco-FT/IR-460 Plus). The absence of calcium oxide traces in the HA phase was checked by the phenolphthalein test.

Figure 1.

X-ray diffraction analysis of the hydroxyapatite (HA) particles manufactured. TCP: tricalcium phosphate.

After calcination, powders were ground for 48 hours to break up agglomerates formed during the thermal treatment and reduce the powder by grinding to its ultimate particle size (around 1 µm).

Methods

Dispersion of hydroxyapatite in alginate aqueous solution

Different concentrations of aqueous sodium alginate solutions were prepared to determine the maximum solubility of the biopolymer in water. The critical transition concentration from the solution to gel state was determined. In the gel state the sodium alginate could not easily infiltrate the nonwoven porous fibrous structure by capillary uptake to coat the inner nonwoven fibers. A total of 3 g/L of alginate solution was the maximum concentration allowing complete solubilization of the sodium alginate without the appearance of any viscous gel.

HA particles are insoluble in water, but when sodium alginate was added to water, the HA particles became homogenously dispersed in the aqueous solution of alginate. Stable, homogeneous and cloudy dispersions of HA particles were obtained using 3 g/L of sodium alginate aqueous solution. The higher the percentage of HA particles used, the cloudier the alginate/HA aqueous dispersions were (Figure 2). Ultrasound sonication (30 minutes at room temperature) was also used to enable a homogeneous distribution of HA in the aqueous sodium alginate solution.

Figure 2.

Stable and homogeneous dispersions formed using three different concentrations of hydroxyapatite (HA; 0.01%, 0.1% and 0.2%) in 3 g/L of aqueous sodium alginate solution.

The dispersion’s stability and homogeneity were assessed by monitoring the behavior of the HA particles when the dispersions were left at rest for 30 minutes. Above 0.2% HA, the sedimentation of HA particles and phase separation occurred. Three different dispersions of HA (0.01%, 0.1% and 0.2%) in 3 g/L of alginate aqueous solution were prepared and were used to functionalize PLA nonwovens.

Surface functionalization of PLA fibers in nonwoven webs

The padding procedure was used to functionalize the PLA nonwovens. Padding consists of dipping of the nonwoven in the aqueous solution of polymer (chitosan or alginate), followed by removal of excess solution by squeezing the nonwoven through two rolling cylinders (see Figure 3).

Figure 3.

Padding method used for the stepwise deposition of chitosan followed by sodium alginate loaded with hydroxyapatite (HA) on poly(lactic acid) (PLA) nonwoven fibers.

Indeed, any unfixed released chitosan or alginate made the conductivity (µs/cm) of the rinsing water increase.

When the PLA nonwoven was padded with the aqueous alginate solution only, the WCA of the PLA nonwoven decreased from 123° to about 20°. However, there was continual release of alginate in rinsing waters, and after removal of all alginate, the WCA of the PLA nonwoven increased back to 120°. This indicated complete removal of alginate polymer from the PLA fiber surface. Electronic repulsion between the negatively charged alginate polymer and the negatively charged PLA would explain this result.

Thus, the layer-by-layer deposition method was used to functionalize the PLA nonwovens, first with a polycationic chitosan polymer before deposition of the anionic sodium alginate polymer. More precisely, the PLA nonwoven was padded first with aqueous chitosan solution (3 g/L) at pH 5, followed by rinsing and then a second padding with an aqueous solution of sodium alginate polymer (with or without HA) – see Figures 3 and 4. The padded sample was washed to remove all unfixed alginate.

Generally, one or two rinsings (5 minutes each) were sufficient enough for removal of all unfixed alginate or chitosan. For all layer-by-layer depositions, three successive washings in distilled water were carried after deposition of chitosan and alginate (without or with HA).

Physico-chemical characterization of functionalized nonwovens

Water contact angle

For measuring WCAs greater than 90°, the sessile drop method using “Digidrop” from GBX Instrument (France) was used.

However, for lower contact angles (<90°), the water drop was absorbed by the nonwoven porous structure. So a more precise method, developed in our previous work called the wicking test, was performed to calculate the WCA as well as the capillary uptake of various nonwovens. A rectangular nonwoven connected to a tensiometer at the weighing position was brought into contact with water placed in a container. On immediate contact, a meniscus weight (W_m) was measured. The WCA of the outer nonwoven membrane surface was calculated using equation (1)

W_{m} x g = γ_{L} xcos θ x p

(1)

where W_m is the meniscus weight (g), p is the sample perimeter in contact with the liquid (mm), g is 9.81 g s⁻², γ_L is the surface tension of water (mN/m) and θ is the WCA (°).

Capillary uptake due to water inflow inside the nonwoven by capillarity was also measured. Whereas the WCA is a measure of hydrophilicity of the outer membrane, capillary uptake is an indicator of hydrophilicity of the inner nonwoven fiber surfaces. At the end of 3 minutes, the nonwoven sample was separated from the water surface, and the weight of water entrapped inside the nonwoven structure by capillarity (W_c) is read directly on the screen of the tensiometer. Experiments were repeated five times for each sample.

More details are given in our previous paper.²⁴

Zeta potential

The surface zeta potential was measured by streaming potential measurement using a Zetacad equipment at 25℃. A total of 0.001 mol L⁻¹ of KCl electrolyte solution was used. A total of 1 g of fibrous PLA nonwoven was maintained in a cell while the electrolyte was forced to flow through the membrane at varying pressures. Before any zeta potential measurement, the sample was maintained in the electrolyte solution for 24 hours in order to reach equilibrium before making the measurement itself. Five measurements were carried out on each sample for pH values of the electrolyte solution varying from 3 to 10 by adding drops of solution HCl or KOH with a concentration of 0.1 mol L⁻¹. More details are described in our previous paper.²⁵

Air permeability of functionalized PLA nonwovens

Air permeability measurements of the fibrous PLA nonwovens were carried out to ensure that layer-by-layer deposition of biopolymers did not block the pores, which would otherwise hinder the infiltration of the biological solution and cells through the inner part of the nonwoven. This test was performed on 10 samples for each functionalized PLA nonwoven adapted from the standard test ISO 9237 using a Textest FX 3300 instrument at a fixed pressure drop of 50 Pa.

The measurements were carried out in dry and wet states. In the wet state, each fibrous scaffold was placed in water for 1 hour and then transferred into a spinner, which allowed removal of excess water in the pores using centrifugal forces. The air permeability tests were then performed. This test is also a measure of the scaffold porosity.

AFM analysis

Investigation using a “Nanoscope III” from Digital Instrument was carried out for AFM imaging in the Tapping mode. The Tapping mode tips the “Budget sensor” from the “Nanoandmore”, of length 125 µm, made of a monolithic silicon probe with an aluminum reflex coating and with resonance frequency of 300 kHz were used.

Tapping mode imaging was carried out in ambient air, and images were plane-fitted.

AFM imaging was carried out on five different fiber samples selected randomly from the PLA nonwoven fibers having been subjected to different treatments. Detailed description of this methodology is described in our previous paper.²⁶

Biological characterization (cell proliferation test)

Different PLA disk samples (virgin and functionalized) were placed in the bottom of 24-well cell culture plates (Costar®, Starlab) after sterilization. Then 7 × 10³ MC3T3-E1 pre-osteoblastic cells were gently seeded in each well. The wells without a disk sample only filled with cell suspension were served as positive control. The duration of cell culture was 3 and 6 days without renewal of the culture medium. After the incubation periods, the MC3T3-E1 cells were detached by 0.05% trypsin–EDTA (Ethylenediaminetetraacetic acid) solution and then counted with a particle counter (Z1, Coulter Electronics Ltd, UK). Six assays were separately preformed and each assay was in triplicate. The final results were rated as the mean of all assays.

Results

Physico-chemical characterization of functionalized nonwovens

SEM images of nonwovens/fiber surfaces functionalized with the layer-by-layer technique using chitosan and alginate biopolymers

Figure 4 gives a schematic representation of the layer-by-layer application of chitosan and alginate (with or without HA), while Figure 5 shows the SEM images of non-functionalized as well as layer-by-layer deposited chitosan and alginate polymers. Zooming in on the bare PLA fiber surface, some regular scaly-type structures appear on the fiber surface (Figure 5(b)). Since just spun PLA fibers have a smooth cylindrical surface, this would mean that the PLA fiber surface features may be due to the method of making the nonwoven web using high water pressure, which would partially damage the PLA fiber surface.

Figure 4.

Schematic representation of layer-by-layer deposition of chitosan and alginate (with or without hydroxyapatite (HA)) on poly(lactic acid) (PLA) nonwoven fibers.

Figure 5.

Scanning electron microscopy images of the nonwoven webs and of the fiber surface before and after functionalization with chitosan and chitosan+alginate. PLA: poly(lactic acid).

After coating with the chitosan, the scaly structures are no longer visible (Figure 5(c)). However, after application of the layer of sodium alginate, a smooth transparent film seems to cover the fiber surface (see Figure 5(d)). Surface corrugations underneath the alginate film are detected. For some nonwovens where no rinsing was carried out after functionalizing with sodium alginate, thin films of alginate film seem to partially block the pores of the nonwoven (see Figure 5(d)). After successive washing this pore blocking film was removed, as confirmed also by microscopy and air permeability results (Table 1).

Table 1.

Wettability, zeta potential and air permeability results of the poly(lactic acid) (PLA) nonwovens at different steps of layer-by-layer deposition

	Water contact angle WCA (°)	Capillary uptake (mg)	Zeta potential (mv) at pH 7.4	Air permeability l/m²/s (when dry)	Air permeability l/m²/s (when wet)	Porosity (%) (dry)
PLA	123 ± 2^a	0	−73 ± 3	1500 ± 10	1500 ± 9	94 ± 2
PLA-chitosan	130 ± 3^a	0	−16 ± 2	1480 ± 15	1480 ± 10	92 ± 1
PLA-chitosan-alginate	62 ± 5	680 ± 10	−36 ± 2	1340 ± 20	1030 ± 20	83 ± 1
PLA-chitosan-alginate-HA 0.01%	55 ± 2	750 ± 11	−53 ± 3	1210 ± 10	1200 ± 8	75 ± 2
PLA-chitosan-alginate-HA 0.1%	55 ± 3	800 ± 10	−53 ± 2	1220 ± 12	1100 ± 10	76 ± 3
PLA-chitosan-alginate-HA 0.2%	55 ± 2	750 ± 15	−53 ± 3	1260 ± 10	1260 ± 7	79 ± 1

Water contact angle measured by Digidrop.

HA: hydroxyapatite.

AFM images of the PLA surface functionalized by the HA/alginate composite

Alginate aqueous solution loaded with one of the three different percentages of HA (0.01%, 0.1% and 0.2%) was used to pad chitosan-coated nonwoven PLA webs. Figures 6(a)–(d) show more detailed AFM topographical images of the functionalized PLA fiber surface. The PLA surface coverage by HA particles seems to depend on the percentage of HA used. The higher the percentage of HA used, the better the PLA fiber surface coverage with HA particles. With 0.01 % of HA, less than 50% of the PLA surface is covered with HA particles. With 0.2% HA, surface coverage with HA is the highest and HA particles are more uniformly distributed. Also, the average size of the HA particles is around 2 µm × 500 nm. With 0.1 % HA, the PLA surface seem to be covered with two different sized HA particles: (1) 2 µm × 0.5 µm and (2) 4 µm × 2 µm.

Figure 6.

Topographical images obtained by atomic force microscopy in the Tapping mode of poly(lactic acid) (PLA) nonwoven fiber surfaces functionalized without or with hydroxyapatite (HA) using chitosan and alginate polymers. (a) PLA -chitosan –alginate, (b) PLA -chitosan –alginate-0.01%HA, (c) PLA -chitosan –alginate-0.1%HA and (d) PLA -chitosan –alginate-0.2%HA.

Zeta potential measurements

Zeta potential arises due to ions in the outer layer around the fiber surface. These ions are representative of the surface charge in direct contact with the aqueous electrolyte solution. The zeta potential values of uncoated and coated nonwoven PLA decrease as a function of the electrolyte solution pH values (see Figure 7). The zeta potential values of uncoated PLA are all negative. The chitosan-coated nonwovens have positive potential values for pH < 7.4, reaching +40 mV at pH 3, and slightly negative potential values for pH = 7.4, reaching −40 mV at pH 8. After application of sodium alginate, zeta potential values vary from −10 mV at pH 3 to around −40 mV at pH 8.

Figure 7.

Streaming zeta potential values of functionalized poly(lactic acid) (PLA) nonwovens as a function of pH. WCA: water contact angle.

The negative charges of the PLA are due to carboxylic groups present at the chain ends of the PLA polymer. Indeed, as pH increases the PLA surface becomes more negatively charged due to the ionization of carboxylic acid groups into carboxylate COO– groups.

The positive charges of the chitosan-coated PLA nonwovens are attributed to the amino groups in chitosan that acquire positive surface charges in an acidic solution by protonation (i.e. from −NH₂ to −NH₃⁺). Ionic interactions between the protonated amino groups (NH₃⁺) from the chitosan coating and the deprotonated carboxylic groups (COO⁻) groups from the PLA lead to an overall zero potential at pH 7.4. At pH > 7.4, the potential zeta values become negative but the values are all above those of the PLA fiber surface, confirming an overall coverage of the PLA surface by the chitosan coating.

When sodium alginate solution is padded over the chitosan-coated PLA, the negatively charged COO⁻ groups in sodium alginate lead to an overall negative charge, but zeta potential values are far less negative than those of the PLA surface.

At a biological medium pH (7.8), the zeta potential of the PLA nonwoven functionalized with the successive layer-by-layer technique using chitosan and alginate is around −35 mV.

For the nonwovens with immobilized HA, at pH 7.8 the zeta potential decreases to −53 mV, whatever the percentage of HA used (see Table 1).

Wettability, zeta potential and air permeability results (see Table 1)

The PLA nonwoven is hydrophobic with a WCA of around 120° (measured using Digidrop) and with null capillary uptake value. This observation is also made for nonwoven PET subjected to the hydro- entanglement method. The WCA value is very high compared to flat PLA or PET films, which normally have a WCA of 78°. Nonwoven surface rigidity due to entangled fiber structures influences the WCA value measurement (see also Behary et al.²⁵).

After padding with chitosan, the nonwoven becomes more hydrophobic, reaching a WCA of 130°. Capillary uptake is also null for the chitosan-coated PLA nonwoven. Padding of the chitosan-coated PLA nonwoven with sodium alginate solution leads to a hydrophilic nonwoven with the WCA decreasing to 62°, and capillary uptake increasing from 0 to 680 mg. The presence of HA particles in the alginate film increases further the hydrophilicity of the PLA nonwovens. WCA lowers down to 53° and capillary uptake increases to around 800 mg with 0.1% of HA.

After functionalization, a slight reduction in air permeability occurs in the dry state for the PLA nonwoven functionalized with chitosan/alginate without HA. As the percentage of HA increases, there is very little change in air permeability. However, when air permeability values of the dry and wet nonwovens are compared, a further decrease in air permeability is measured in the wet state, most probably because the alginate and chitosan films at the PLA surface absorb water, swell and, thus, reduce the PLA nonwoven pore diameter.

Zeta potential and wettability results show that overall, HA particles immobilized with the layer-by-layer technique using chitosan/alginate layers increase the fiber surface wettability of PLA. Moreover, zeta potential values are more negative (−53 mV) in the presence of HA compared to the chitosan/alginate film without HA (−36 mV). These results also imply that the HA particles are not entirely covered with the alginate film: they protrude out of the alginate film and are exposed. HA (Ca₁₀(PO₄)₆(OH)₂) particles link to the free COO− groups of the alginate polymer via the calcium ions, while the hydroxyl and phosphate groups contribute to increased negative zeta potential. Moreover, HA particles are hydrophilic in nature²⁷ and contribute to an increase in hydrophilicity of the nonwovens.

Final discussion on physico-chemical characterization of functionalized nonwoven poly(lactic acid)

Although the HA powders synthesized were around 1 µm, the HA microparticles form agglomerates. Ultrasound sonication enabled uniform and stable dispersion of HA particles in sodium alginate solution. The chitosan coating cationized the PLA fiber surface, providing good adhesion of the HA-loaded anionic alginate coating. HA was almost homogeneously distributed at the PLA fiber surfaces without reducing considerably the average pore size, as confirmed by air permeability values of the nonwovens.

Functionalization using layer-by-layer deposition of chitosan and alginate with HA leads to a negatively charged, rough (due to HA particles) and hydrophilic surface.

Biological characterization (cell culture-adhesion and proliferation)

Figure 8 shows the number of rat pre-osteoblast MC3T3-E1 cells counted after 3 and 6 days of culture for a seeding with 7 × 10³ MC3T3-E1 pre-osteoblastic cells.

Figure 8.

In vitro proliferation of rat pre-osteoblasts MC3T3-E1 cells. The number of cells counted after 3 and 6 days of culture for a seeding with 7 × 10³ MC3T3-E1 pre-osteoblastic cells.

Although all cells added during initial seeding did not survive, part of them adhered and proliferated on the composite three-dimensional-PLA-chitosan-alginate (HA) porous fibrous scaffolds.

When nonwovens functionalized with different proportions of HA are compared, the number of cells adhering after 6 days increases as the percentage of HA increases. Thus, with 0.2% HA, almost 1 × 10⁴ pre-osteoblastic cells adhered after 3 days of culture, which doubled to almost 2 × 10⁴ after 6 days.

Discussion

Important scaffold parameters involve the control of the scaffold degradation rate, the non-cytotoxicity of scaffold degradation products,²⁸ and also the scaffold surface properties. In the selection and fabrication of the suitable biomaterial, pore size, pore structure, surface topography and chemistry, biodegradation, hydrophilicit, and surface charge and energy are important factors.^29,30

A PLA nonwoven structure based on microfibers was successfully functionalized with HA particles using layer-by-layer deposition of chitosan followed by sodium alginate loaded with HA particles, without considerable reduction in pore size, as confirmed by air permeability measurements. Ultrasound sonication enabled uniform and stable dispersion of HA particles in sodium alginate solution.

Indeed, the three-dimensional biodegradable PLA/chitosan/alginate/HA composite surfaces are more hydrophilic than the PLA fiber surface and have a high (microsized) surface roughness with negative zeta potential values. HA particles are not entirely covered with the alginate film: they protrude out of the alginate film and are exposed.

Biological tests show that the higher loading with HA is, the higher the bone cell number is after 6 days of culture.

Further studies must be performed to investigate cell morphology and distribution, as well as new extracellular matrix (ECM) formation in the interstitial space between fibers.

Conclusions

In this work, a three-dimensional porous fibrous composite PLA/HA scaffold was developed by immobilizing HA micro particles on the PLA fiber surface using layer-by-layer deposition of chitosan and alginate polymers, by a padding method. HA particles were properly dispersed in the sodium alginate aqueous solution allowing their immobilization at the PLA surface.

AFM and SEM images, as well as zeta potential measurements, showed surface modifications resulting from the deposition of chitosan and of HA-loaded alginate and confirmed the overall coverage of PLA nonwoven fiber surfaces with the layer-by-layer chitosan and alginate layers. The composite nonwoven structure provides a scaffold that allows a certain degree of pre-osteoblast cell proliferation, which increases as the content of HA increases. Our work also shows that PLA microfibers of diameter 30 µm and nonwoven pore dimensions reaching 230 µm can be effective in promoting bone cell migration and proliferation.

Footnotes

Acknowledgements

This work was realized within the framework of Fédération des Biomatériaux du Nord – Pas-de-Calais. The authors also would like to thank Francois Dassonville and Christian Catel for their kind help.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

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