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BioNanoMaterials

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Volume 15, Issue 1-2

Issues

Fabrication and characterization of electrospun polylactide/β-tricalcium phosphate hybrid meshes for potential applications in hard tissue repair

Loredana Tammaro
  • Department of Industrial Engineering, University of Salerno, Via Ponte Don Melillo 1, Fisciano, I-84084 (SA), Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Vittoria Vittoria
  • Department of Industrial Engineering, University of Salerno, Via Ponte Don Melillo 1, Fisciano, I-84084 (SA), Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Ralf Wyrwa / Jürgen Weisser / Birgitt Beer / Susanne Thein / Matthias Schnabelrauch
Published Online: 2014-08-05 | DOI: https://doi.org/10.1515/bnm-2014-0001

Abstract

Non-woven hybrid meshes based on poly(l-lactide-co-d,l-lactide) (dl-PLA) and β-tricalcium phosphate (β-TCP) were fabricated and comprehensively characterized. Stock suspensions of β-TCP powder in dl-PLA acetone solutions were used for the electrospinning process. Structure, morphology and thermal properties of the electrospun samples were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The electrospun hybrid materials show a micro-composite structure, and a morphology characterized by a three-dimensional porous fibrous mesh with randomly distributed fibers possessing average fiber diameters between 680 and 970 nm, uniform thickness along the fibers and beads structure only for higher β-TCP concentration electrospun samples. Both pure PLA and hybrid non-woven meshes exhibit a good thermal stability and a continuous degradation in simulated body fluid medium. A live/dead staining viability assay using MC3T3-E1 preosteoblasts reveals the excellent cytocompatibility of the fabricated non-wovens. Enhanced alkaline phosphatase (AP) activity of MC3T3-E1 cells during culture on the dl-PLA and the composite non-woven meshes demonstrates their potential for applications in hard tissue repair.

This article offers supplementary material which is provided at the end of the article.

Keywords: biodegradable composites; bone repair; cytocompatibility; electrospinning; nanofibers; tissue engineering

Introduction

During the last decade electrospinning has emerged as an efficient technique to produce nano and micro diameter-sized fibers possessing a broad range of potential applications in the field of tissue regeneration [1–8]. A polymer solution, when electrostatically charged, can produce non-woven polymeric fibers in different size ranges by controlling the physical properties of the polymer solution and the spinning process conditions. Therefore, many technical parameters can be modulated to obtain desired fiber dimensions and designed physical properties. Moreover this technology possesses various inherent features, such as the simplicity and inexpensive nature of the set up, the ability to produce continuous fibers of various materials and the potential scale up of the process, which makes it currently the preferred and most popular technique for the fabrication of ultrafine fibers [9, 10].

A further advantage of this processing technique is its ability to fabricate scaffold structures of high and inter-connective porosity similar to the natural extracellular matrix. Because biodegradability is an essential material requirement in tissue engineering, numerous potentially biodegradable polymers including poly(α-hydroxy acids) and their copolymers [11], polyanhydrides [12], collagen [13], gelatin [14], chitosan [15], or silk [16] have been used to fabricate scaffolds for cell culture. Numerous studies have shown that different types of cells including osteoblasts [17], chondrocytes [18], fibroblasts [19], keratinocytes [20], or cardiomyocytes [21] are able to adhere and proliferate well when cultured on those electrospun scaffold materials.

Although synthetic polymers like polylactones are easily processed to form complex shapes and structures, often they lack bioactive function, i.e., initial protein adsorption and cell adhesion as well as strong interfacial bonding of the implant to living bone tissue due to their intrinsic hydrophobicity and low surface energy and the impairment of the formation of a biologically active apatite layer on the polymer surface [22, 23]. Therefore, recent attempts have been undertaken to combine biodegradable polymers with inorganic materials mimicking the mineral bone phase. Bioactive ceramics or glasses, especially different calcium phosphates like hydroxyapatite (HA, micro- and nano-sized), carbonated, calcium-deficient HA (CDHA), β-tricalcium phosphate (β-TCP), amorphous TCP (aTCP), dicalcium phosphate anhydrate (DCPA) magnesium phosphate or particular bioactive glass have been used to produce novel composites fiber meshes for tissue engineering and other biomedical applications [24]. Experiments have proven that the mechanical as well as biological performance of those bioactive hybrid materials can be efficiently controlled through using different particulate inorganic fillers and also through varying the amount of filler materials in the composite [24, 25]. Examples for recently reported hybrid material combinations are poly(lactide-co-glycolide) (PLGA)/aTCP [26], poly(D,L-lactide) (PDLL)/aTCP [27], polylactide/DCPA [28], PDLL/HA [29] or polylactide/CDHA [30].

Both dense and porous composites based on β-TCP have been demonstrated to show excellent performance in the physiological environment and possess several advantages over other calcium phosphates especially with regard to bioresorption [31–36]. In contrast only a few papers have been published on the fabrication of electrospun biodegradable hybrid fiber meshes containing β-TCP as inorganic component. In the previously reported work poly(ε-caprolactone) [37–39] or poly(L-lactide) [40], which are known to exhibit a rather slow rate of degradation have been used to prepare β-TCP or β-TCP/HA-containing hybrid compositions. There are no systematic studies available which elaborate optimum process parameters for electrospinning of polymer mixtures containing β-TCP particulates with varying β-TCP content. Also the influence of different β-TCP contents in the hybrid material on the resulting properties of the fiber meshes has not been reported in detail for different biodegradable polymers.

In our work we were interested in a hybrid material with swift and continuing degradability and good acceptance for bone forming cells. We therefore have chosen poly(l-lactide-co-d,l-lactide) (dl-PLA) as a well-known degradable and approved polymer and established a reliable and efficient process to provide electrospun dl-PLA scaffolds containing β-TCP as a bone-like additive. We evaluated the effects of basic process parameters including solution concentration, solution flow rate, needle-collector distance, and applied voltage on the characteristics of the electrospun dl-PLA/β-TCP hybrid meshes. The structure, morphology and thermal behavior of the electrospun composite fibers were analyzed by X-ray diffraction, scanning electron microscopy, thermogravimetry and differential scanning calorimetry, respectively. The biodegradation behavior was monitored in vitro. The cytocompatibility of dl-PLA/β-TCP meshes and their capability to stimulate alkaline phosphatase (AP) formation of MC3T3-E1 preosteoblastic cells seeded on these meshes were examined.

Results and discussion

Influence of relevant process parameters on the homogeneity of the resulting electrospun mats

Electrospun meshes were fabricated using two different concentrations of dl-PLA in acetone, 2.5 and 5% w/w. The concentration of the polymeric solution influences the spinning of fibers and controls the morphology. If the concentration of dl-PLA in acetone was increased from 2.5 to 5% w/w the polymer has not reached complete dissolution in the solvent anymore and as a consequence pieces of fibrous polymeric material are formed as visible in the SEM images (see Figure S1 in the Supporting Information). As a consequence all experiments using different dl-PLA/β-TCP compositions have been performed using 2.5% w/w polymeric solutions.

Increasing the needle-collector distance, the morphology was changed from a beaded fiber to uniform fiber structure. At a distance of 5 cm in some screen areas besides fiber formation film-like structures (marked with arrows) are visible, probably due to the small needle-screen distance not allowing the solvent evaporation. Increasing the distance from 5 to 20 cm the amount of beads in the sample decreases, and uniform and bead-free fibers were obtained at a distance of 20 cm (see Figure S2 in the Supporting Information). These results are in good agreement with findings recently published for the preparation of electrospun meshes from several synthetic polymers like polystyrene [41].

A series of experiments were carried out varying the solution flow rate from 0.5 to 5.0 mL/h. The electrospun fibers of pure dl-PLA were again examined by SEM. The micrographs for the different fibrous samples investigated are shown in Figure 1. Fibers produced with the highest and also the lowest flow rate (1.0 vs. 5.0 mL/h) tended to form spindle-like fibers as depicted in Figure 1A and D. In contrast, a uniform fiber shape with a similar thickness along the fiber was found for meshes where intermediate values of flow-rate (e.g., 2.0 and 3.0 mL/h) have been applied. Similar results have been recently reported by J Du [42].

SEM images of electrospun dl-PLA solution at flow rate of (A) 1.0 mL/h, (B) 2.0 mL/h, (C) 3.0 mL/h and (D) 5.0 mL/h.
Figure 1

SEM images of electrospun dl-PLA solution at flow rate of (A) 1.0 mL/h, (B) 2.0 mL/h, (C) 3.0 mL/h and (D) 5.0 mL/h.

The different process parameters were optimized for all β-TCP containing compositions (dl-PLA/P5, dl-PLA/P10, dl-PLA/P20). These results are summarized in Table 1 including the average diameter of the resulting fibers.

Table 1

Average diameter of electrospun fibers with different β-TCP content under the used processing conditions (polymer solution concentration 2.5% w/w, voltage 20 kV, needle-collector distance 20 cm).

Morphology and structure of dl-PLA and dl-PLA/Px fibers

Representative SEM photographs and the fiber diameter distribution of the electrospun dl-PLA and the β-TCP-containing dl-PLA composites are shown in Figure 2. About 100 fibers were tested in order to determine the diameter distribution. For neat dl-PLA the fibers diameter ranged between 650 and 850 nm. The addition of β-TCP powders into dl-PLA solution did not appear to hinder the electrospinning process, but slightly and continuously increased the average fiber diameter (see Table 1). Kruskal-Wallis one way analysis of variance (ANOVA) on ranks was used to check differences in the median values. The differences among the groups were greater than would be expected by chance (p=<0.001). Pairwise multiple comparison by Dunn’s method as well as by Tukey test showed the median of all groups were different from each other with statistical significance (p<0.050) with the exception of the difference between dl-PLA/P10 and dl-PLA/P20. Using the less conservative Student-Newman-Keuls method also these groups differed significantly. In the hybrid meshes, when the filler concentration was increased from 5 to 20% w/w, the mesh morphology changed gradually from uniform fiber structure to a poor bead structure. The results indicated that all the electrospun meshes presented a three-dimensional porous fibrous mesh appearance consisting of randomly orientated PLA fibers with similar thickness along the fiber.

SEM images and diameter distributions of dl-PLA, dl-PLA/P5, dl-PLA/P10 and dl-PLA/P20 electrospun fibers (flow rates for the preparation of the meshes are given in Table 1).
Figure 2

SEM images and diameter distributions of dl-PLA, dl-PLA/P5, dl-PLA/P10 and dl-PLA/P20 electrospun fibers (flow rates for the preparation of the meshes are given in Table 1).

XRD patterns of β-TCP powders and hybrid dl-PLA/β-TCP meshes are compared in Figure 3. The original dl-PLA phase shows a low crystallinity, with two main crystalline peaks appearing at around 16.5° and 18.7° of 2θ. At variance, the broad pattern of the non-woven mesh can be attributed to the reflection of amorphous dl-PLA, indicating that the evaporation of the solvent during the electrospinning process is very rapid not allowing the polymer crystallization. All patterns of electrospun hybrid samples showed the broad pattern associated with the amorphous dl-PLA together with some characteristic peaks of the β-TCP phase, although of different relative intensity. Moreover comparing the samples with lower (5% w/w) and higher (20% w/w) content of β-TCP we observe that the X-ray reflections in dl-PLA/P20 are even less intense, indicating a partially amorphous incorporation of the inorganic material into the fibers.

X-ray diffraction profiles of (a) β-TCP powder, (b) dl-PLA powder, (c) dl-PLA fibers, (d) dl-PLA/P5 fibers and (e) dl-PLA/P20 fibers.
Figure 3

X-ray diffraction profiles of (a) β-TCP powder, (b) dl-PLA powder, (c) dl-PLA fibers, (d) dl-PLA/P5 fibers and (e) dl-PLA/P20 fibers.

Thermal properties

The thermogravimetric analysis was performed for investigating both the degradation temperature and the inorganic component content into the fibers. The thermal decomposition up to 1000°C in air of the electrospun dl-PLA and its hybrid meshes are shown in Figure 4. dl-PLA thermally degrades to volatile products in the temperature range of 250–450°C. The weight loss profiles of all hybrids are very similar each other being characterized by one important weight-loss stage above 300°C. The introduction of different amounts of inorganic filler slightly anticipates the degradation temperature of the electrospun dl-PLA which occurs at about 342°C. The weight residual corresponds to the inorganic loading of the meshes.

Thermal degradation profiles of dl-PLA and dl-PLA/Px hybrid fibers under air flow.
Figure 4

Thermal degradation profiles of dl-PLA and dl-PLA/Px hybrid fibers under air flow.

To investigate the effect of the electrospinning process and the inorganic powder introduction on the thermodynamic behavior, DSC traces of electrospun dl-PLA meshes with and without β-TCP were recorded (Figure 5). As already found by XRD we do not observe any endothermic peak due to the polymer crystallinity, but only the glass transition is showing an enthalpic content which decreases on increasing the inorganic percentage in the sample. As shown in Table 2 lower glass transition temperatures (Tg) are observed for the electrospun non-woven composite than pure polymer fibers. By adding the inorganic filler into the electrospun polymer fibers, the small particles seem not perfectly to interact with the polymer chains which may result in an increase of the free volume thus leading to a lower Tg [43].

Calorimetric curves (DSC) of electrospun fibers in nitrogen atmosphere.
Figure 5

Calorimetric curves (DSC) of electrospun fibers in nitrogen atmosphere.

Table 2

Glass transition temperatures (Tg) of dl-PLA and dl-PLA/Px hybrid samples.

In vitro degradation

Degradation of polylactides under physiological conditions depends on various parameters including polymer composition and structure, surface-volume ratio, surface morphology, and the incorporation of additives and filler materials. Normally, measurement of mass loss over time is an appropriate method to assess the degradation rate of dense biomaterials, but this method is not well suited for electrospun materials due to their low mass and high surface area. Therefore an enzymatic lactate assay based on the enzyme lactate dehydrogenase was established to determine the amount of released l- and d-lactate from the samples. In Figure 6, the degradation of dl-PLA, and the dl-PLA/Px composites are shown based on the cumulative total lactate (sum of released l- and d-lactate).

Cumulative determination of total lactate (sum of l- and d-lactate) released during degradation of dl-PLA and dl-PLA/Px composite fiber meshes in SBF medium at 37°C.
Figure 6

Cumulative determination of total lactate (sum of l- and d-lactate) released during degradation of dl-PLA and dl-PLA/Px composite fiber meshes in SBF medium at 37°C.

Both the dl-PLA and dl-PLA/Px non-woven meshes show a continuous degradation over the studied time interval of 12 weeks. Comparing l- and d-lactate release graphs of dl-PLA and dl-PLA/Px, respectively (data not shown), it was found that the different monomers are released in accordance to their molar contents (85% of l-lactate and 15% of d-lactate) in the starting polymer. Only minor differences in the degradation rate were found between the pure dl-PLA non-woven and the β-TCP-containing composite fibers containing 5 and 20% w/w of β-TCP, respectively. Interestingly the composite fibers containing 10% w/w of β-TCP exhibited a somewhat lower rate of in vitro degradation. It seems that several parameters contribute to this different behavior. On one side the degradation of the electrospun meshes correlates mostly with the fiber diameters shown in Figure 2. In case of higher amounts of thinner fibers of 450–750 nm (dl-PLA, dl-PLA/P5) the release of lactate is increased compared to fiber meshes with a narrow fiber distribution of about 850 nm (dl-PLA/P10). An exception represents the dl-PLA/P20 mesh which degrades faster than dl-PLA/P10 despite possessing thicker fibers. It is assumed that due to the high amount of β-TCP (20% w/w) the homogeneity within the fiber composite formed during the electrospinning process is lower compared to compositions with lower β-TCP content. This might result in faster leaching processes of β-TCP particles. Further studies are necessary to explain this phenomenon more in detail.

Biological properties

The FDA/GelRed™ viability assay was performed to assess the cytocompatibility of the produced non-woven meshes. Viable cells were labelled using fluorescein diacetate, whereas dead cell nuclei were identified by GelRed™ uptake. After cultivating MC3T3-E1 preosteoblasts for up to 21 days on dl-PLA, dl-PLA/P5, dl-PLA/P10, and dl-PLA/P20 electrospun meshes no signs of cytotoxicity could be detected. Fluorescence micrographs (Figure 7) revealed that the cell density after 22 days was consistently higher than after 1 day; hence, the cells proliferate. A visible difference between the three fiber meshes could only be detected in the first 8 days of culture where on the β-TCP-containing meshes (dl-PLA/P5, dl-PLA/P10, dl-PLA/P20) a considerably higher cell density was observed compared to the dl-PLA mesh. However, after 15 days no differences in cell density could be found anymore between the meshes. After 22 days, the cells had not only well adhered to the sample surfaces but had also grown into a continuous, dense cell layer on the fiber mesh surfaces demonstrating the excellent cytocompatibility of these materials.

Viability assessment of MC3T3-E1 preosteoblasts cultured on electrospun dl-PLA and dl-PLA/Px [green fluorescending vital cells shown for dl-PLA, dl-PLA/P5, dl-PLA/P10, and dl-PLA/P20; at 1, 8, 15, and 22 days; red fluorescending dead cell nuclei shown for the materials after 21 days (last row)].
Figure 7

Viability assessment of MC3T3-E1 preosteoblasts cultured on electrospun dl-PLA and dl-PLA/Px [green fluorescending vital cells shown for dl-PLA, dl-PLA/P5, dl-PLA/P10, and dl-PLA/P20; at 1, 8, 15, and 22 days; red fluorescending dead cell nuclei shown for the materials after 21 days (last row)].

The higher cell densities of β-TCP containing meshes at day 8 might be attributed to positive effects of calcium released from the fibers. Although pure dl-PLA overall is assessed as cytocompatible it has been shown that released substances may decrease DNA synthesis and thus proliferation of BALB 3T3 cells in vitro [44]. Since cells growing at electrospun meshes additionally do not find optimal conditions for cell adhesion at this substrate, proliferation might be affected also by this parameter. The presence of calcium, which is a very important factor in cell adhesion, should improve the situation. Moreover, lactic acid slowly released from dl-PLA should be in equilibrium with its calcium salt and therefore possibly shows other cellular effects.

AP formation, as an indicator of cell osteogenic potential, was investigated during culture of MC3T3-E1 preosteoblasts. A quantitative determination of AP activity of MC3T3-E1 preosteoblasts was performed after 7, 14, and 21 days of culture. Values for the specific AP activity which is total AP activity related to the cellular protein amount are shown in Figure 8.

Specific alkaline phosphatase activity of MC3T3-E1 cells cultured on dl-PLA, dl-PLA/P5, dl-PLA/P10, and dl-PLA/P20 meshes for different culture times.
Figure 8

Specific alkaline phosphatase activity of MC3T3-E1 cells cultured on dl-PLA, dl-PLA/P5, dl-PLA/P10, and dl-PLA/P20 meshes for different culture times.

Preosteoblasts grown at glass control samples showed clearly lower specific AP values than the cells grown at the meshes (<50% of the lowest mesh sample) independent from measurement time. Nevertheless, a low but continuous increase by about 20–25% was observed at the glass reference with time. Preosteoblasts growing at dl-PLA and at dl-PLA/P20 possessed a very constant specific AP activity independent from time, but at dl-PLA/P20 the level was about 9% higher. This level was reached also at dl-PLA/P5 after 14 and 21 days, although osteoblasts of this group had the lowest level of specific AP after 7 days within the different groups of meshes. A clear increase was also observed at dl-PLA/P10 exceeding the levels of dl-PLA after 14 and 21 days most clearly. Statistical analysis by two way repeated measures ANOVA (the two factors were factor A: the type of mesh sample including glass control, and factor B: the different time points) showed that the differences between the mesh types (factor A) were statistically significant (p=<0.001). But this was verified in pairwise multiple comparison only for the differences between the meshes and the control. The difference in the mean values of the time points (factor B) was not great enough to exclude the difference was just due to random sampling. The effect of different mesh types did not depend on what time point was captured. Summing up, addition of β-TCP stabilized the osteogenic differentiation of MC3T3-E1 cells but the sample size was too small (n=3) to substantiate the statistical significance.

Staining of AP positive cells grown on different non-woven meshes using the HNPP/Fast Red TR method revealed that characteristic formation of AP positive cell clusters was sustained independent from the content of calcium phosphate in the meshes. Staining results presented in Figure 9, which shows the total MC3T3-E1 preosteoblasts visualized by DAPI staining of their nuclei (blue spots) and the part of AP positive cells coloured reddish by the HNPP/Fast Red TR method, illustrate that after 22 days of culture the β-TCP-containing meshes possess also distinct AP activity similar to the pure dl-PLA mesh. AP activity detected by staining seemed to be relatively low at the pure dl-PLA mesh at day 8 but this fluorescence staining did not allow an exact quantification. Nevertheless, the images suggest the decrease of specific AP activities was in general the result of a somewhat higher proliferation of AP negative cells.

Fluorescence images illustrating DAPI and HNPP/Fast Red TR staining (superposition) of MC3T3-E1 cells cultured on dl-PLA, dl-PLA/P5, dl-PLA/P10 and dl-PLA/P20 meshes after 8 days (upper row) and 22 days (lower row) of culture.
Figure 9

Fluorescence images illustrating DAPI and HNPP/Fast Red TR staining (superposition) of MC3T3-E1 cells cultured on dl-PLA, dl-PLA/P5, dl-PLA/P10 and dl-PLA/P20 meshes after 8 days (upper row) and 22 days (lower row) of culture.

Overall, the excellent cytocompatibility of the prepared hybrid meshes and their capability to promote cell differentiations indicate their potential for applications in bone tissue engineering.

Conclusions

Optimum electrospinning conditions to obtain non-woven meshes of poly(l-lactide-co-d,l-lactide) (dl-PLA) charged with different concentrations of β-TCP powders were studied and set up. The structural analysis showed that the polymer matrix is amorphous and its glass transition temperature slightly decreases with increasing the β-TCP concentration due to the free volume created by the inorganic filler. The presence of the filler does not change much the fiber dimensions and their mean distribution. The morphology is characterized by three-dimensional porous fibers distributed randomly with average diameters ranging between 680 and 970 nm, uniform thickness along the fibers and beads structure only for higher β-TCP concentration in the electrospun samples. Both pure PLA and hybrid meshes are degraded in simulated body fluid medium. A live/dead staining viability assay using MC3T3-E1 preosteoblasts reveals the excellent cytocompatibility of the fabricated hybrid meshes. Enhanced AP activity of MC3T3-E1 preosteoblast cells during culture on the PLA and the non-woven hybrids demonstrate their potential as scaffold materials for applications in hard tissue repair.

Materials and methods

Materials

dl-PLA (l-lactide:d,l-lactide composition=67:33–73:27 molar ratio, Mw=1.35×106 g/mol, Resomer 708) was purchased from Boehringer Ingelheim (Germany). β-Tricalcium phosphate (β-TCP) powders (sauter diameter: 2.70 μm) and acetone were purchased from Sigma-Aldrich (Italy). All reagents and solvent were used as purchased without further purification.

Electrospinning of dl-PLA and dl-PLA/Px

dl-PLA solutions of 2.5 and 5% w/w of polymer were prepared by dissolving a measured amount of dl-PLA in acetone under vigorous stirring at room temperature for 3 h. For preparing the hybrid meshes (denoted as dl-PLA/Px, with x representative of β-TCP weight percentage) the corresponding amounts of β-TCP were added to the acetone polymer solution (2.5% w/w total polymer concentration) under the same previously mentioned conditions.

A laboratory electrospinning machine was used to fabricate the meshes. The dl-PLA and dl-PLA/Px solutions were placed into a syringe (5 mL) connected to a needle of an inner diameter of 0.8 mm. The solutions were electrospun by applying a variable positive voltage (from 20 to 35 kV) to the needle using a high voltage power supplier purchased from Gamma High Voltage Research, Ormond, FL. The distance from the syringe tip to the collector plate was investigated at variable values from 5 to 20 cm, and the polymer solutions were loaded at variable feed rate from 0.1 to 5.0 mL/h. The collected electrospun meshes were stored in vacuum for 24 h to remove any solvent residue.

Thermal analysis (TGA, DSC)

Thermoanalytical characterizations (TGA) were performed by a Mettler TC-10 thermobalance operating at 5°C/min heating rate, under air flow, from 30 to 1000°C. Degradation temperature (Td) is reported as the midpoint of the degradation step.

Differential Scanning Calorimetry (DSC) measurements were carried out using a Mettler DSC 822/400 thermal analyzer instrument having sub-ambient capability. About 2–3 mg sample was placed in an aluminium pan and heated at a rate of 10°C/min from –40 to 150°C in a nitrogen atmosphere.

Structural and morphological analysis (XRD, SEM)

X-ray powder diffraction (XRD) data were collected using an automatic Bruker diffractometer (equipped with a continuous scan attachment and a proportional counter), with the nickel filtered Cu Kα radiation (λ=1.54050 Å) and operating at 40 kV and 40 mA. The diffraction scans were recorded at 2θ=5–40°, step size 0.05°, time per step 3 s.

To analyze the morphology of the electropun structures and to measure the diameter of the fibers forming the structure all samples were sputter coated with gold (Agar Automatic Sputter Coater Mod. B7341, Stansted, UK) at 30 mA for 180 s and analyzed by a scanning electron microscope (SEM Mod. LEO 420, Assing, Italy).

In vitro degradation assay

About 80 mg of the electrospun materials (dl-PLA and dl-PLA/Px) was used to determine the in vitro degradation. All samples were exposed to 5 mL of simulated body fluid (SBF) medium of pH 7.4 in a thermostatic incubator at 37°C over 6 weeks. Every week half of the medium was withdrawn and the SBF in the vessel was replenished up to 5 mL. The degree of degradation was monitored by the l-lactate sample concentration using an enzymatic l-lactate assay (R-Biopharm, Darmstadt, Germany) according to the manufacturer’s protocol.

Cell seeding experiments

Electrospun specimens (dl-PLA, dl-PLA/P5, and dl-PLA/P10) were cut to squarish pieces of about 20×20 mm. Square microscopic slides of 10×10 mm were covered with the samples exposing the meshes to the top. The overlaying rims were folded below the slides. Three replicates of each material for every time point were employed. These samples were transferred to 24 well cell culture plates (Greiner, Frickenhausen, Germany) and disinfected with about 1 mL of 70% aqueous ethanol for 30 min. Thereafter the ethanol was removed by medium exchange two times against phosphate buffered saline (PBS, consisting of 137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4) and once against cell culture medium. The samples were seeded with 1 mL of a suspension of MC3T3-E1 cells (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) at a density of 25,000 cells/cm2. The cells were cultured with osteogenesis inducing medium. (alpha medium supplemented with 10% FCS, 2 mM L-Ala-GlN, 50 U/mL penicillin, 50 μg/mL streptomycin, all from Biochrom, Berlin, Germany, 0.1 μM dexamethasone, and 10 mM beta-glycerophosphate) at 37°C under a 5% CO2 atmosphere. The medium was renewed every 2 days.

Cytocompatibility testing

The influence of the different meshes on viability and proliferation of MC3T3-E1 preosteoblasts was examined by using the fluorescein diacetate (FDA)/GelRed™ (VWR International, Darmstadt, Germany) viability assay. The viability staining followed conventional FDA/ethidium bromide (or propidium iodide) protocol, but ethidium bromide was substituted by the less toxic GelRed™. GelRed™ showed identical results in previous live/dead staining experiments as ethidium bromide (own observation). Like ethidium bromide it is excluded from DNA-staining [45] by intact cell membranes (cf. product information). After 1, 7, 14, and 21 days, the culture medium was replaced by PBS, the samples were placed onto microscopic slides, overlaid with equal amounts of two-fold concentrated staining solution (0.030 mg/mL fluorescein diacetate, 2×GelRed™ in PBS; GelRed™ stock reagent was purchased 10,000×), and evaluated microscopically. Green and red fluorescence were monitored after 1 min using fluorescence microscopy (Axiotech, Zeiss, Jena, Germany). Photomicrographs were recorded using a CCD fluor microscope imager MP 5000 (Intas, Goettingen, Germany). Imaging was supported by Image-ProPlus 5.1 software (Media Cybernetics, Silver Spring, MD).

DAPI and HNPP/Fast Red TR staining

AP positive cells were stained by use of the HNPP fluorescent detection set (Roche Applied Science, Mannheim, Germany) according to the manufacturer instructions. Briefly, the cells grown on supporting materials were fixed for 10 min by 70% ethanol in PBS, washed for 10 min in washing buffer (100 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) and for 10 min in detection buffer (100 mM Tris/HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2). The detection buffer was replaced with 0.05 mL staining solution (0.01% w/v 2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate (HNPP), 0.025% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride (Fast Red TR) in detection buffer and the cells were incubated for 30 min at room temperature in the staining solution. The reaction was stopped by washing in PBS. Cell nuclei were stained by addition of 2 μg/mL 4′, 6-diamidino-2-phenylindole (DAPI) in PBS and incubation for 5 min in the dark. Red fluorescent HNP (dephosphorylated HNPP)/Fast Red TR precepitate (excitation 553 nm, emission 584 nm) were analysed by use of fluorescence microscopy (see above) using Zeiss filter set 14, fluorescence of DAPI staining (excitation 358 nm, emission 461 nm) was observed with filter set 02. Both images were taken seperately and merged during image processing. All the cell nuclei fluoresced blue while only AP positive cells appeared red fluorescent.

AP activity

For the measurement of AP activity the samples were prepared as in cell seeding experiments. Square microscopic slides of 10×10 mm made of glass served as control and reference. At 7, 14, and 21 days the samples were withdrawn, transferred to a new 24 well cell culture plate and washed two times with 1 mL of PBS by medium exchange. PBS was discarded and the cells were lysed with 0.1 mL of lysis reagent (CytoBuster™, Merck Biosciences, Darmstadt, Germany). Lysates were resuspended by the pipette, transferred to 1.5 mL reaction tubes. Undissolved material was spun down at 14,000 rpm for 5 min. Aliquots were taken from the supernatant for the measurement of AP activity and colorimetric assay of total protein. For the measurement of AP 5 μL lysate were mixed with 0.1 mL chemiluminescence substrate solution (CDP-StarR, Roche-Diagnostics, Mannheim, Germany) in a white 96 well microplate (Greiner) and luminescence was measured after 20 min in a multiwell plate photometer enabling luminescence measurements (Genios Pro, Tecan, Germany).

Protein content was measured by the Micro BCA method (Thermo Scientific, Perbio Science, Bonn, Germany), according to the manufacturer’s instructions. Bovine serum albumin was used as a standard.

Statistics

Statistical analysis were performed using SigmaPlot 11.2 software (Systat Software, Erkrath, Germany) in order to test differences in the mean diameters of fibers and mean values of AP measurements on statistical significance. In case of fiber diameters, one hundred measurement values were acquired from each group by image evaluation. Kruskal-Wallis one way analysis of variance on ranks was performed since normality test (Shapiro-Wilk) failed (p<0.050). Pairwise multiple comparison procedures have been calculated according to Dunn’s method, to Tukey test and to Student-Newman-Keuls method. For statistical analysis of AP measurements two way repeated measures ANOVA was applied, including normality test (Shapiro-Wilk) and equal variance test at a significance level of 0.050. Post hoc tests for multiple comparisons were run with predetermined alpha values of 0.050 by the Holm-Sidak method.

Acknowledgments

We acknowledge the support of the European Commission through the ArtiVasc 3D project under the Seventh Framework Programme (Grant Agreement no 263416).

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Supplemental Material

The online version of this article (DOI: 10.1515/bnm-2014-0001) offers supplementary material, available to authorized users.

About the article

Corresponding author: Matthias Schnabelrauch, Biomaterials Department, INNOVENT e. V., Pruessingstrasse 27B, D-07745 Jena, Germany, Phone: +49 (0)3641 282512, Fax: +49 (0)3641 282530, E-mail:


Received: 2014-01-14

Accepted: 2014-05-26

Published Online: 2014-08-05

Published in Print: 2014-09-01


Citation Information: BioNanoMaterials, Volume 15, Issue 1-2, Pages 9–20, ISSN (Online) 2193-066X, ISSN (Print) 2193-0651, DOI: https://doi.org/10.1515/bnm-2014-0001.

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