Introduction

Synthetic biodegradable polymers have been studied extensively over the past decades as potential scaffold materials for tissue engineering. Such materials must fulfill numerous requirements including bioresorbability and biocompatibility, support of cell proliferation and adhesion, and must have mechanical properties appropriate to surrounding tissue while being permeable to nutrients and oxygen. The properties of the materials must therefore be tailored to suit each application.

Our research group is experienced in the synthesis and development of functionalized aliphatic polyesters as scaffold materials. For example, poly(l-lactide) and poly(ε-caprolactone) with double bonds in the polymer backbone have been successfully prepared. These functional groups have further been oxidized to epoxides and cross-linked to obtain grafted polymers and hydrogels with improved mechanical properties [1, 2]. The aliphatic copolyesters have been shown to be promising materials in tissue engineering. For example Idris et al. [3] and Dånmark et al. [4] have shown that scaffolds formed from copolymers of l-lactide and ɛ-caprolaction as well as 1,5-dioxepan-2-one (DXO) permit significant cell attachment and proliferation of human osteoblast-like cells. Further, it has been shown that the bioactivity of these scaffolds was improved by adding bone morphogenetic protein-2 (BMP-2) and by increasing hydrophilicity. We now seek to improve the results of these scaffolds by bioengineering scaffolds that sequentially deliver specific growth factors in order to obtain optimal bone tissue regeneration.

Hydrogels appear to be promising materials in tissue engineering because of their hydrophilic character and a structure similar to the native extracellular matrix (ECM) [5, 6]. The field of regenerative medicine has been particularly interested in hydrogels formed as the result of external stimuli, the so-called in situ-forming hydrogels, which transition from solution to a gel in response to either solvent exchange, UV irradiation, or change in pH or temperature. The last of these in situ-forming hydrogels, also called thermosensitive or thermo-reversible hydrogels, are particularly interesting for tissue engineering because they can be injected into scaffolds or directly into tissues to be used as scaffolds. This has many advantages compared to other materials because surgical procedures can be avoided and they can easily adapt to the shape of the damaged area. In addition they can also be prepared without use of organic solvents, and cells and growth factors can easily be incorporated [7].

Many different thermosensitive hydrogels have been synthesized and tested for medical applications, e.g., poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAM-co-AM) [8], poloxamers [9], block copolymers of poly(ethylene oxide) (PEO) and poly(l-lactide) (PLLA), and block copolymers of PLLA, poly(ethylene glycol) (PEG), and polyglycolide (PGA) [10, 11]. However, none of these are particularly good materials for tissue engineering because they are either nondegradable or degrade/resorb too quickly in vivo. The sol–gel transition temperatures of block copolymers of PLLA, PGA, and PEG are also difficult to modify and still preserve the sol–gel transition temperature which is due to the sensitivity of the block length of the PEG segment. To overcome this problem Jeong et al. [12] synthesized grafted copolymers of PLLA, PGA, and PEG (PLGA-g-PEG) which were porous, thermo-reversible, biodegradable, and persisted in vivo up to 3 months. Further studies also showed that the hydrogel was biocompatible, nontoxic, and that by changing the length of the hydrophobic and hydrophilic segments of PLGA-g-PEG the sol–gel transition temperature could be adjusted [13, 14].

The porous 3D scaffolds and the injectable hydrogels described above are both good materials for tissue engineering. Here we describe our efforts to develop scaffolds that combine the advantages of both these materials, giving a material which potentially could be used for sequential release of desired compounds to improve tissue regeneration.

Materials and methods

Materials

l-Lactide (LLA) and glycolide (GA) (Boehringer Ingelheim, Germany) were used as received. Epichlorohydrin, poly(ethylene glycol) methyl ether (MPEG; M n = 500 g/mol), anhydrous tetrahydrofuran (THF), anhydrous acetonitrile, N,N-diisopropylethylamine (DIPEA), Dulbecco’s modified Eagle Medium (D-MEM), Pluronic F-127, anhydrous magnesium sulfate, and stannous octoate (Sn(Oct2)) were ordered from Sigma-Aldrich, Germany. Hexane, dichloromethane, and chloroform were ordered from Fisher Scientific, USA. Sodium hydroxide and sodium hydrogen carbonate was ordered from VWR, Sweden and phosphate buffered saline solution (0.1 mM, PBS) was ordered from PAA, Austria. Hydroxyapatite (HA) was bought from Riedel-de Häen, Germany.

Methods

Synthesis of poly(l-lactide-co-ε-caprolactone) porous scaffolds

Poly(l-lactide-co-ε-caprolactone) porous scaffolds were prepared as previously described by Dånmark et al. [4].

Synthesis of epoxy-terminated poly(ethylene glycol) methyl ether

Two different methods were used to prepare epoxy-terminated poly(ethylene glycol) methyl ether (EPEG) (Fig. 1). The first method was similar to the one described by Cho et al. [15]. Briefly, 59.5 g of MPEG (M n = 550 g/mol) and 10 g of NaOH were mixed in a 200-mL round flask, to which 10.8 mL of epichlorohydrin was slowly added. The mixture underwent magnetic stirring and was heated at 40 °C for 2 h. The product was then purified by addition of acetonitrile and the excess unreacted NaOH and formed NaCl were separated by filtration. The excess unreacted MPEG and acetonitrile were subsequently distilled off by rotary evaporation.

Fig. 1
figure 1

Reaction scheme for the synthesis of EPEG

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In the second method, 59.5 g of MPEG (M n = 550 g/mol), 10.8 mL of epichlorohydrin, 35 mL DIPEA, and 100 mL THF were mixed in a 200-mL round flask. The mixture was kept in a heated oil bath (60 °C) for 15 h. The reaction was then stopped by the addition of a small amount of 10 wt% solution of citric acid and the THF was removed by rotary evaporation. DIPEA was separated from the product by liquid–liquid extraction. A 10 wt% solution of citric acid was used as extraction solvent and the product was extracted with dichloromethane. The extraction was repeated three times followed by addition of sodium bicarbonate to remove acidic residues from the solution. The sodium bicarbonate and sodium chloride formed as side products were then removed by filtration and liquid–liquid extraction using deionized water as extraction solvent. To remove traces of water, magnesium sulfate was added to the product. When a complete absorption was achieved the product was separated from the drying agent by filtration and dichloromethane was removed by rotary evaporation.

Synthesis of poly(l-lactide-co-glycolide) grafted with poly(ethylene glycol)

PLGA-g-PEG was synthesized with a Sn(Oct)2-based, one-step ring-opening polymerization of LLA, GA, and EPEG (Fig. 2). The reaction was prepared in a silanized round-bottomed flask under nitrogen atmosphere inside a dry box (Mbraun MB 150B-G-I). Polymerization was initiated by immersing the flask into a thermostatic oil bath. The reaction temperature and time were set at 120 °C and 48 h, respectively. The product was purified by dissolution in chloroform and precipitation in cold hexane.

Fig. 2
figure 2

Reaction scheme for the synthesis of PLGA-g-PEG

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Preparation of thermosensitive hydrogel with and without hydroxyapatite

A 30-wt% portion of PLGA-g-PEG was added to 10-mL vials and dissolved in D-MEM and PBS at 20 °C (room temperature). The co-oligomers formed a homogenous solution after approximately 3 h. Hydrogels containing HA were prepared in the same way as described above. A 10-wt% portion of HA was added to the solution after a homogeneous solution was formed. The mixture was kept at room temperature under magnetic stirring until the solution was homogeneously dispersed.

Nuclear magnetic resonance (NMR)

Characterization and co-oligomer composition of EPEG and PLGA-g-PEG were determined by NMR. The spectra were obtained by using a Bruker Advance DPX-400 NMR operating at 400.13 MHz; 1 mL deuterochloroform (CDCl3), was used to dissolve approximately 5 mg and 100 mg of the sample for 1H NMR and 13C NMR, respectively. The samples were prepared in NMR sample tubes (diameter = 5 mm).

Size exclusion chromatography (SEC)

The molecular weight and polydispersity index (PDI) of EPEG were determined by SEC, using chloroform as mobile phase (4 mg of the sample was dissolved in 8 mL of the solvent and filtered into 2-mL vials).

The samples were analyzed with a Verotech PL-GPC 50 Plus system equipped with two PLgel 5 μm MIXED-D (300 × 7.5 mm) columns, an autosampler, and a PL-RI detector from Varian (Munich, Germany). The Cirrus™ GPC Software program was used to process data. Chloroform was used as the mobile phase and the eluent ran at 1.0 mL/min with a temperature of 30 °C. Polystyrene standards were used for calibration with a molecular weight distribution range of 162–371,100 g/mol and corrections for the flow rate fluctuations were made using toluene as an internal standard.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

MALDI-TOF-MS was performed using a Bruker UltraFlex system and used to calculate the molecular weight and PDI of EPEG and PLGA-g-PEG. The analysis was performed with a SCOUT-MTP ion source in reflector mode equipped with a 337-nm nitrogen laser. The acceleration voltage and reflector voltage were 25 kV and 26.3 kV, respectively, and the analysis was run in positive ion mode. Super 2,5-dihydroxybenzoic acid (SDHB) was used as matrix and NaI was used as cationizing agent. The sample was dissolved in a mixture of dichloromethane and methanol (50:50). The sandwich method was used as sample preparation technique wherein 0.5 μL of a 10 mg/mL matrix solution was first added followed by 0.5 μL of a 1 mg/mL sample solution secondly and then 0.5 μL of a 1 mg/mL salt solution. The spectra obtained from the analysis were based on an accumulation of 10 spectra, every spectrum of which was taken with 200 laser shots at 10 different spots. The laser power was set to 40–50 % and the mass-to-charge ratio range was m/z 550 to 3,000.

Test tube inversion method

The sol–gel transition temperature of the PLGA-g-PEG hydrogel was determined by a test tube inversion method. A 30-wt% portion of the co-oligomer was dissolved in PBS and D-MEM and 0.5 mL was added to 2-mL vials. The vials were immersed in 37 ± 1 °C thermostatic oil baths and kept there for 1 min. The vial was then taken out, inverted, and the flow of the oligomer solution was investigated. When there was no visual macroscopic flow the solution was considered a gel.

pH measurement

To determine the pH of the co-oligomer solutions a VWR symphony meter SB70P equipped with a Biotrode pH electrode from Hamilton was used. The electrode had a pH measuring range of 0–14 and the reference electrolyte was a Protelyte electrolyte. The instrument was calibrated with a two-point calibration using buffer solutions at pH 4 and 7.

Functional lifetime

To determine the functional life of PLGA-PEG, 30 wt% and 10 wt% oligomer solutions in D-MEM and PBS with and without 10 wt% of HA were prepared. The solutions were added to 2-mL vials and put in an incubator at 37 °C. The samples were taken out every 24 h and the flow of the solutions was investigated. As long as no flow was observed the samples were considered to be functional. We used 30-wt% and 10-wt% oligomer solutions of Pluronic F-127 as reference.

Preparation of hierarchical porous scaffolds

A particulate salt-leached poly(l-lactide-co-ε-caprolactone) (PLCL) scaffold (diameter 10 mm, height 5 mm) was placed in a 2-mL vial. A 30-wt% PLGA-g-PEG in D-MEM solution was added to the vial and the mixture was subjected to ultrasonication for 2 min. The composite was then heated in an oil bath at 37 °C for 1 min. The gelation was tested by the test tube inversion method as described above. To determine that a successful hierarchical porous scaffold (i.e., a scaffold which has two components with different resorption times) had been formed, the cross section of the scaffold and the flow of the hydrogel solution were analyzed. To analyze the cross section, the scaffold was frozen in liquid nitrogen and taken out by gently breaking the glass of the vial. A cross section was then made with a scalpel to ensure that the gel had penetrated the PLCL scaffold. The surface was analyzed with an 8-megapixel iSight camera.

Results and discussion

Synthesis of EPEG

We first optimized the synthesis route for EPEG. The route that is most often reported uses NaOH as an activator of MPEG [12]. The advantage of this strong and unhindered base is that the reaction time is short (2 h); however, undesired side products that are hard to remove are formed. To prevent this, NaOH was replaced by DIPEA, which is a strong but hindered base. As DIPEA is not as strong a base as NaOH the reaction time was approximately 10 times longer, but because it is a poor nucleophile, no unwanted side products were formed and the purification was both easier and more efficient. EPEG, prepared using DIPEA, was characterized by 1H NMR, 13C NMR, 2D-HSQC, and DEPT NMR. The characterization of EPEG is shown in Fig. 3.

Fig. 3
figure 3

1H NMR (left) and 13C NMR (right) spectra of EPEG with the corresponding peaks presented in the table. The samples were analysed in CDCl3

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The characteristic peaks of the epoxy group protons (δ = 2.91, 2.71, and 3.26) as well as the peaks originating from MPEG (δ = 3.65 and 3.39) in the 1H NMR spectrum show that a successful synthesis of EPEG was obtained. It should be pointed out that a full confirmation of the synthesis could not be made because the expected changes in shift of the protons and carbon (from the release of a chloride atom and binding to the MPEG) overlap with the peaks from the PEG unit. A comparison with 1H NMR and 13C NMR spectra from a mixture of epichlorohydrin and MPEG also confirmed this, showing identical spectra where no difference between the reacted and unreacted reactants can be seen (the spectra are presented in supplementary data, Figs. S1 and S2). To prove that EPEG had been synthesized, the NMR analysis was combined with two other characterization methods, MALDI-TOF-MS and SEC, the results of which are presented in Table 1 and Fig. S3.

Table 1 Molecular weight of EPEG determined by SEC and MALDI
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Comparison of the theoretical molecular weight of EPEG with the SEC and MALDI-TOF-MS results demonstrates that the synthesis of EPEG was successful. The SEC and MALDI-TOF-MS results show a higher molecular weight than the theoretical molecular weight but the molecular weight was nonetheless higher than the molecular weight of MPEG and epichlorohydrin, which strongly suggests that they reacted with each other and formed EPEG.

Synthesis of PLGA-g-PEG, a thermosensitive hydrogel

We next mapped the reaction between LLA, GA, and EPEG in detail. As the goal was to synthesize oligomers with few repeating units, the monomer feed ratio and the corresponding product composition were critical.

The peak originating from the PEG unit of PLGA-g-PEG (3.63 ppm) has been used in past studies to determine the amount of EPEG in the product. The problem with this is that the number of protons representing this peak is not constant and we therefore used the peak that originated from the protons of the methyl group on the grafted PEG (3.39 ppm), which represents a defined number of protons.

As shown in Table 2, the conversion of both LLA and GA was high in all reactions. The changes in the feed correspond well to the changes in the composition of the oligomers, using GA as an internal reference. For both LLA and EPEG the amount in the feed was higher compared to the amount in the oligomer.

Table 2 Composition and monomer conversion of PLGA-g-PEG
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Thermosensitive hydrogels with a sol–gel transition temperature of 37 °C were achieved for samples 1–3. In sample 4 the feed of EPEG was doubled which resulted in an oligomer with twice as much EPEG as samples 1–3. This resulted in a more hydrophilic co-oligomer that did not form a gel at 37 °C. The viscosity of sample 4 was also decreased from a sticky paste to an easily flowing solution. When the feed of EPEG was decreased to 0.25 in sample 5, the amount of EPEG in the oligomer was decreased by 50 % compared to samples 1–3. A more hydrophobic co-oligomer with high viscosity was thus obtained and, due to the increase in hydrophobicity, it was insoluble in water-based solutions.

The molecular weights of the oligomers were determined by MALDI-TOF-MS. The mass spectra were studied in Flex analysis and the M n and PDI were determined using Polytools (Bruker Daltonics). For samples 1–3, Polytools identified oligomer chains with M n between 900 and 1,100 g/mol and PDI between 1.01 and 1.02. Average values of M n and PDI were calculated from these results and are shown in Table 2.

Structure analysis of PLGA-g-PEG

The structure of the oligomers plays a central role in sol–gel behavior and because the molecular weight is low it was very important to know the exact monomer sequence and relate that to gelling properties. A careful structural analysis of the oligomers was determined by 1H NMR, 13C NMR, 2D-HSQC NMR, and DEPT NMR. The possible configurations of the oligomer are illustrated in Figs. 4 and 5, and the origins of the peaks from these structures are shown in Fig. 6.

Fig. 4
figure 4

Molecular configurations of PLGA-g-PEG synthesized by ROP of LLA, GA and EMPEG

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Fig. 5
figure 5

Molecular configurations of PLGA-MPEG synthesized by ROP of LLA, GA and MPEG

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Fig. 6
figure 6

1H NMR of PLGA-g-PEG with the corresponding peaks presented in the table. The sample was analysed in CDCl3

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From the 1H NMR spectrum it can be seen that an oligomer including LLA, GA, and EPEG was obtained (5.17/4.95–4.58/3.39 ppm). The strong poly(l-lactide) and weak LLA monomer peaks confirm a high LLA conversion and, in addition, there is no peak between 3 and 2.5 ppm which confirms a high conversion of EPEG. To prove that the monomers have been linked together and that it was not merely homopolymerization that occurred, the peaks were compared with those in the 1H NMR spectrum from poly(l-lactide-co-glycolide) (PLGA) and poly(l-lactide-co-glycerol) (PLLA-glycerol). The coupling between PLLA and glycerol is identical to the coupling between PLLA and EPEG and was therefore chosen as the reference. It was not possible to obtain a good 1H NMR reference for the coupling between GA and EPEG because GA is not soluble in chloroform. However, the structures of LLA and GA are similar and it is likely that the coupling would be located in the same area. The comparison of the spectra is presented in Fig. 7, with PLGA-g-PEG at the bottom (A), PLLA-glycerol in the middle (B), and PLGA at the top (C). It is clearly seen that the peaks at 4.95 to 4.58 in the PLGA-g-PEG spectrum originate from the linkage between LLA and GA, whereas the peaks between 4.44 and 4.25 originate from the couplings between LLA and EPEG. In the 1H NMR spectrum of PLGA there are peaks in the area between 4.95 and 4.58 which are not present in the PLLA-glycerol spectrum. These peaks can only come from the coupling between LLA and GA because homopolymers of GA cannot be seen in chloroform and homopolymers of LLA appear at 5.17 ppm. The peak originating from PLLA can be seen in all spectra, indicating that homopolymers of LLA were present in the product as well as in PLGA and PLLA-glycerol. The peaks between 4.44 and 4.25 in PLGA-g-PEG can only be seen in the PLLA-glycerol spectrum which suggests that they originate from the linkage between PLLA and EPEG. This finding is also consistent with earlier structural analysis of PLGA [16] and PLLA-EPEG [15].

Fig. 7
figure 7

A comparison of the 1H NMR of PLGA-g-PEG (A), PLLA-glycerol (B), and PLGA (C). The samples were analysed in CDCl3

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Although there is coupling between the LLA and EPEG it was not clear if the EPEG was added as an end group, as methoxy-PEG (MPEG), or grafted within the chain. 13C NMR analysis of PLGA-g-PEG was therefore performed to identify the coupling between the monomers in PLGA-g-PEG and to determine if PLGA-MPEG also had been formed. The couplings were studied by 13C NMR, 2D-HSQC NMR, and DEPT NMR, comparing PLGA, PLLA-glycerol, poly(l-lactide-co-poly(ethylene glycol) methyl ether) (PLLA-MPEG), and poly(glycolide-co-poly(ethylene glycol) methyl ether) (PGA-MPEG). The couplings identified are shown in Table 3 and 13C NMR of PLGA-g-PEG and the reference spectra are shown in Fig. 8. From the 13C NMR, 2D-HSQC NMR, and DEPT NMR spectra of PLLA-glycerol, PLLA-MPEG, and PGA-MPEG (C, D, and E), it can be concluded that in addition to PLGA-g-PEG, PLGA-MPEG was also formed. Peaks number 2 and 3 are both present in the PLGA-g-PEG spectra and according to the DEPT NMR and 2D-HSQC NMR these are CH2 carbons which correlate with the protons between 4.44 and 4.25 ppm (i.e., the protons which correspond to the coupling between GA-EPEG/MPEG or LLA-EPEG/MPEG). The peak to the left of peak number 2 is also a CH2 carbon, but it correlates with the protons in GA. It therefore seems logical that a single EPEG or MPEG was located as an end group on the oligomers resulting in very similar structures. This would explain the missing coupling between PLLA-EPEG at 62.5 ppm and the suggestion is also supported by the missing EPEG-EPEG coupling at 63 ppm in the product spectra.

Table 3 Couplings between the monomers in PLGA-g-PEG
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Fig. 8
figure 8

13C NMR spectrum of PLGA-g-PEG (A) and comparison with PLGA (B), PLLA-glycerol (C), PLLA-MPEG (D), PGA-MPEG (E), and Poly(epoxy-terminated ethylene glycol methyl ether) (PEPEG) (F)

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The amounts of couplings (C) were calculated from the intensities (I) of the 13C NMR peaks of the carbons originating from the couplings between the monomers of PLGA-g-PEG. The peak originating from the coupling between LLA-LLA was used as reference. Since the EPEG-LLA/GA couplings were located in the same area as those of MPEG-LLA/GA, a total amount for these couplings was determined as shown below:

$$ {C}_{LLA- GA}={I}_{LLA- GA}/{I}_{LLA- LLA} $$

The amount of couplings between LLA, GA, MPEG, and EPEG are shown in Table 4. Samples 1–3 formed stable gels within 1 min at 37 °C, whereas samples 4 and 5, which had twice and one half as much EPEG, respectively, did not form gels at 37 °C. It can be seen that the amount of EPEG in the samples corresponds to the amount of coupling to EPEG/MPEG in the samples. Sample 4 had the highest amount of couplings between LLA-EPEG/MPEG and GA-EPEG/MPEG and sample 5 the lowest. The reactivity ratio of LLA towards GA was lower than towards itself and it can also be seen that the reactivity of LLA towards GA was higher than towards EPEG/MPEG. The amounts of coupling between GA and EPEG/MPEG are lower than between LLA and MPEG, which seems reasonable because the literature suggests that the reactivity ratio of GA is higher than that of LLA. From these results it can be concluded that the co-oligomer was random.

Table 4 Amount of couplings between the l-lactide, glycolide, EPEG, and MPEG
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Results from the 1H NMR and 13C NMR strongly indicate that both PLGA-g-PEG and PLGA-MPEG were present in the product. Since the oligomers contain one EPEG or MPEG unit located as an end group, the possibility cannot be excluded that the synthesized PLGA-g-PEG oligomer might be mixed with PLGA-MPEG. This would mean that the gelation properties were not only related to PLGA-g-PEG but to a mixture of PLGA-g-PEG and PLGA-MPEG.

Sol–gel transition temperature

It has been shown that the sol–gel transition temperature can be varied with different compositions of the monomers [17]. Longer hydrophilic segments in the copolymer increase the sol–gel transition temperature and longer hydrophobic segments decrease the sol–gel transition temperature. The sol–gel transition temperature of the oligomer was determined by the test tube inversion method. The oligomer solution was heated in a thermostatic oil bath at 37 °C for 1 min and the flow of the solution was determined. To prepare an oligomer with a sol–gel transition temperature of 37 °C the amounts of LLA and GA were kept constant and the amount of EPEG was varied. A successful hydrogel was obtained when the molar ratio between the monomers was 2.53/1/0.49 (LLA/GA/EPEG). The gelation is illustrated in Fig. 9.

Fig. 9
figure 9

30 wt% PLGA-g-PEG oligomer solution at room temperature (a) after being heated at 37 °C (b)

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Consistent with earlier studies, the sol–gel transition temperature of sample number 4 (which had twice the amount of EPEG) was higher than those of samples 1–3 and did not form a gel at 37 °C. It was not possible to determine the sol–gel transition temperature for sample 5 because it was not possible to dissolve it in buffer solution. The addition of 10 wt% HA to the polymer solutions (with the same composition as samples 1–3) did not influence the sol–gel transition temperature. Gels were successfully obtained at 37 °C which was also consistent with earlier research [18].

pH

The use of low molecular weight products affects the pH and the pH of the gel was therefore measured over time. The 30 wt% oligomer solutions were prepared using two different media, PBS and D-MEM, which both have a pH of 7.4. Four different samples were prepared, two where only the medium was added and two where medium and 10 wt% HA were added. The results are shown in Fig. 10.

Fig. 10
figure 10

pH of PLGA-g-PEG in PBS (black squares), D-MEM (black circles), PBS + HA (white squares), and D-MEM + HA (white circles)

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The highest pH was obtained when D-MEM was used as solvent. The pH decreased with time and was likely caused by the low molecular weight oligomers and thus related to the number of acidic end groups. Similar results have been reported previously, where the acidic nature of PLGA low molecular weight oligomers caused a decrease in pH [19]. The pH in PBS was much lower and it was obvious that the composition of the different media had a great effect on the final pH. PBS buffers are based on solutions containing sodium phosphate and sodium chloride. The phosphate helps stabilize the pH of the solution around 7 but because phosphate has three pK a values (pK a1 2.12, pK a2 7.21, pK a3 12.67) it can also be stabilized at lower pH. The PBS buffer was not strong enough to maintain the pH at 7 when the PLGA-g-PEG oligomer was added. As a result of the acidic end groups it instead stabilized at a lower pH closer to pK a1.

It has been shown that the pH of the oligomer solutions is increased by the addition of hydroxyapatite [18]. It is believed that the hydroxyapatite helps neutralize the acidic environment caused by the co-oligomers and it is also an important ingredient in bone formation. When hydroxyapatite was added to the PLGA-g-PEG solutions the pH was increased as expected. The greatest increase was obtained in the PLGA-g-PEG PBS solution, where the pH increased from 3.8 to 5.8. When hydroxyapatite was added to the PLGA-g-PEG D-MEM solution, only a small increase in pH was observed. However, the solution was stabilized at a higher pH for a longer time. The pH was stabilized at 6 which could be interesting for an in vivo cell environment.

Functional life

The results of the functional life evaluation are shown in Fig. 11. Pluronic F-127 (12,700 g/mol) was used as reference. The functional life of PLGA-g-PEG oligomer was shorter than that reported for PLGA-g-PEG in another publication [14] because of the difference in molecular weight. The PLGA-g-PEG oligomer has a lower molecular weight compared to earlier research on PLGA-g-PEG, which causes faster degradation and resorption. The functional life was under 1 month in both PBS and D-MEM, which makes it a promising material for sequential release in PLCL scaffolds (because the degradation time is shorter than that of the PLCL scaffolds)[20]. It could also be seen that the functional lifetimes of the D-MEM samples were slightly longer than those of the PBS samples. When HA was added to the PLGA-g-PEG oligomers the functional life of samples was increased even more (up to 4 months when D-MEM was used as medium). In addition, HA improved the gel properties of PLGA-g-PEG. Without the addition of HA, a 10 wt% solution did not form a gel, but a stable gel was formed and lasted up to 26 days when HA was added. This stabilizing effect was not observed for Pluronic F-127. It was not even possible to dissolve 30 wt% Pluronic F-127 with HA in PBS or D-MEM. The stabilizing effect and extended functional life of the D-MEM samples with HA may be caused by the increased formation of hydrogen bonds between the OH groups in HA and D-MEM and the carbonyl group in LLA and GA of PLGA-g-PEG. Similar results were reported by Zhou et al. [21] and Lin et al. [18] where Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) show increased hydrogen bond interactions when HA was added to PLLA.

Fig. 11
figure 11

The functional lifetime of 10- and 30 wt% oligomer solutions of PLGA-g-PEG. Samples were prepared in two different solvents (PBS and D-MEM with and without 10 wt% HA) and analysed by using the test tube inversion method. The samples were considered as functional as long as no macroscopic flow was observed

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Analysis of hierarchical porous scaffolds

A combination of this hydrogel with porous 3D scaffolds (a fast-releasing system combined with a slow-releasing system) would be beneficial for bone tissue regeneration, among other possible uses. Analysis of the hierarchical porous scaffold is shown in Figs. 12 and 13. The results from the test tube inversion study showed that the PLGA-g-PEG successfully formed a gel upon heating at 37 °C for 1 min in the scaffold.

Fig. 12
figure 12

Hierarchical porous scaffolds before and after being heated at 37 °C for 1 min

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Fig. 13
figure 13

Hierarchical porous scaffolds taken out from a vial (a), Cross-section for the scaffold-hydrogel composite (b and c)

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The analysis of the cross section showed that the co-oligomer solution penetrated the scaffold all the way in to the center. The scaffold and hydrogel formed a uniform system so that a sequential release of growth factors should be possible.

Conclusion

Thermosensitive low molecular weight PLGA-g-PEG oligomers were successfully synthesized and injected into a PLCL scaffold forming a hierarchical porous scaffold. By careful analysis of the structure and properties of PLGA-g-PEG a hydrogel was obtained. It formed stable gels within 1 min at 37 °C and had a functional life of approximately 3 weeks. In addition, it was found that the gelation properties were not only related to PLGA-g-PEG but also to PLGA-MPEG. Because of their structural similarities, it was impossible to show with NMR that gelation is due to PLGA-g-PEG alone. Addition of HA to the PLGA-g-PEG yielded improved stability of the gel and an increased pH and functional lifetime, likely due to the increased number of hydrogen bonds between HA and the LLA and GA units. Analysis of this system has increased our understanding of the behavior of low molecular weight PLGA-g-PEG thermosensitive hydrogels which, together with PLCL porous scaffolds, allows for a promising sequential controlled release system for growth factors.