Chemically modified glycosaminoglycan derivatives as building blocks for biomaterial coatings and hydrogels

Starting GAG derivatives for modification

Selective chemical reactions on GAG require suitable conditions such as good miscibility. Especially, the high solution viscosity requires, particularly for native HA, the prior degradation of the GAG down to molecular weights of 500–20 kDa. Retaining the original polymer structure free of undesired, e.g. unsaturated and cytotoxic degradation products is essential. The thermal degradation of HA in an autoclave at 130 °C was identified as suitable degradation process (Kunze et al. 2010). The final molecular weight can be adjusted by choosing the process time resulting in molecular weights from 500 to 6 kDa. Further methods are oxidative degradation, e.g. by ozone (Rother et al. 2015). The reduction of the weight-average molecular weight (M _w) of native HA was found to be depending on the degradation time for both procedures (Table 1).

Sulfation of HA and CS

The biological properties of GAG are massively influenced by the degree of sulfation and the sulfate group distribution within the disaccharide repeating units. In particular, the higher sulfated GAG can only be isolated to a limited extent with structural uniformity from biological sources and they are accessible by total synthesis only at extremely high time expenditure. For this reason, the stepwise and regioselective sulfation of HA and CS is a time- and cost-efficient approach mimicking important biological features of higher-sulfated GAG (Scharnweber et al. 2015). Therefore, numerous synthesis pathways for chemical sulfation of native HA and CS have been developed and were reviewed in the past (Bedini et al. 2017; Schiller et al. 2015).

Presently, complexes of SO₃ with organic amines (trimethylamine, triethylamine, pyridine) or amides (DMF) are mainly used for introducing sulfate groups into GAG under gentle reaction conditions. Here, the GAG are first converted into the corresponding tetrabutylammonium salts, which are dissolved or strongly swollen in aprotic solvents such as DMF. This allows widely homogeneous reaction control.

We optimized a sulfation strategy for the synthesis of sHA derivatives using SO₃-pyridine as sulfating agent in a homogeneous reaction by varying the sulfation time (60 vs. 20 min for the synthesis of high- and low-sulfated hyaluronan, respectively) (Rother et al. 2021) as illustrated in Figure 2.

Figure 2:

Synthesis of high- and low-sulfated HA derivatives using the SO₃-pyridine complex as sulfating agent (Rother et al. 2021). DS_S indicates the average number of sulfate groups per disaccharide repeating unit of GAG.

GAG (meth)acrylates

(Meth)acrylated GAG are versatile components enabling the fabrication of functional coatings (Krieghoff et al. 2019), hydrogels (Rother et al. 2017), and cryogels (Thönes et al. 2017). The chemical syntheses of the HA-based (meth)acrylate derivatives are depicted in Figure 3. Methacrylation was performed by the reaction of HA with methacrylic acid anhydride, whereas acrylate functions were introduced by a phase-transfer-catalyzed reaction using acryloyl chloride in the presence of tetra-n-butyl ammonium fluoride (TBAF) (Rother et al. 2017).

Figure 3:

(Meth)acrylation of hyaluronan (HA) and hyaluronan sulfates (sHA1, sHA1Δ6S).

(Meth)acrylation of all HA derivatives has been performed starting from thermally degraded native materials. GAG-sulfate (meth)acrylates can be analogously synthesized starting from the corresponding sulfated GAG (CS, DS_S: 0.9), low-sulfated HA (sHA 1, DS_S: 0.9–1.0), sulfated at the C-6 position of the GlcA repeating unit, and sHA1Δ6S (DS_S: 1.0–1.4), sulfated only at the secondary OH-positions of C-2′, C-3′, and C-4 but non-sulfated at the C-6 position (Schiller et al. 2015). HA, sHA, and CS could be (meth)acrylated resulting in DS values (DS_AC, DS_MA: average degree of (meth)acrylate groups per repeating unit) ranging from 0.1 to 0.7 (Rother et al. 2017).

As revealed by estimation of the sulfur content, the (meth)acrylation took place without loss of sulfate groups. The molecular weight of both, the non-sulfated and sulfated GAG, was reduced only slightly due to the mild reaction conditions as determined by GPC (Becher et al. 2013).

Crosslinking of GAG (meth)acrylates forming coatings and dimensionally stable hydro- or cryogels occurs by free radical polymerization in the presence of a water-soluble initiation system like ammonium persulfate/N,N,N′,N′-tetramethylethylenediamine (APS/TEMED) or photochemically after addition of a photo-initiator and UV irradiation (Becher et al. 2013).

Collagen/GAG-based coatings

Coating materials with coll/GAG-based artificial extracellular matrices (aECM) including sHA and CS derivatives were first performed for tissue culture polystyrene (reviewed in Scharnweber et al. 2015). The procedure was successively adapted to scaffolds and implants for further in vitro and in vivo studies: For biodegradable, porous poly(l-lactide-co-glycolide) (PLGA) containing one or two types of pores as well as for lactic acid-based (TriLA) scaffolds with different porosity, coatings were performed using syringes and applying slight vacuum. This way, the fibrillogenesis solution containing tropocollagen with and without sHA3 was forced to infiltrate the porous scaffold architecture and entrapped air was removed (Krieghoff et al. 2019; Wojak-Ćwik et al. 2019). Collagen fibrillogenesis with immersed scaffolds occurred over night. The amount of immobilized coating was further enhanced by drying the scaffolds in the remaining aECM solution after transfer to tissue culture plates before subjecting them to in vitro analyses.

To analyze aECM coatings for in vivo bone healing of biodegradable polycaprolactone-co-lactide (PCL) scaffolds, various GAG (HA, CS, sHA3) were included (Förster et al. 2020; Neuber et al. 2019). Here, the scaffolds were only immersed in fibrillogenesis solution with fibrillogenesis taking place over night and lyophilized afterwards.

Acrylated hyaluronan/collagen-based hydrogels

Functional hydrogels for improved wound healing applications have been developed from acrylated hyaluronan (HA-AC) embedding coll type I fibrils into the photo-crosslinked network (Rother et al. 2017, 2019, 2021; Thönes et al. 2019). The aim was to derive defined cellular microenvironments as well as carrier or scavenging systems for growth and inflammatory factors to control cellular responses. CS and sHA derivatives were included into this network either covalently by using low sulfated, acrylated GAG derivatives, i.e. sHA1-AC, sHA1Δ6S-AC and CS-AC (Rother et al. 2017, 2021; Thönes et al. 2019) or as part of a coll-based aECM containing high-sulfated sHA3 (Rother et al. 2019). The latter were incorporated into the bulk of the hydrogel or via aECM-containing HA-based microgels generated in a microfluidic approach.

GAG derivatives

Methods of GAG polysaccharide analysis are limited because powerful methods such as mass spectrometry cannot be applied. Due to the low volatility, it is impossible to transfer the GAG into the gas phase without a significant extent of decomposition. Thus, the most common way to study conventional GAG relies on their depolymerization into oligosaccharides. Although this is possible by oxidation, enzymatic digestion is beneficial, since this is a gentler method. Unfortunately, many enzymes are inhibited by the sulfate residues. Thus, it has to be emphasized that high-sulfated GAG cannot be enzymatically converted into oligosaccharides posing a significant challenge regarding GAG characterization. A detailed, very recent review of GAG analysis is available in Khan et al. (2020). A selection of methods for GAG analysis is illustrated in Figure 4.

Figure 4:

Survey of analytical strategies to characterize native GAG polysaccharides with a moderate sulfate content and chemically sulfated GAG.

Methods suitable for GAG characterization

There are three different parameters, which characterize an unknown GAG:

1. The average molecular weight and the extent of dispersity.

2. The type of disaccharide repeating units (e.g. such as GlcA and GlcNAc).

3. The number and positions of the sulfate residues.

We will focus here on sulfated GAG, which are refractive to enzymatic digestions (Lemmnitzer et al. 2014).

Chromatographic methods

Chromatography is one of the most powerful analytical tools for polysaccharide analysis. A special form of size exclusion chromatography (SEC), gel permeation chromatography (GPC) separates molecules based on their size or, more exactly, their hydrodynamic volume. Separation of the analyte, dissolved in the mobile phase, occurs on a stationary phase containing porous beads. The retention time of analyte molecules depends on their interaction with the pores of the beads in the stationary phase. In the standard equipment, the molecular weight (MW) determination by GPC represents a relative method and requires the use of polysaccharide standards (e.g. dextrans and pullulans). More advanced GPC systems with integrated double or multiple detection allow also absolute MW determination. In addition to the number average and the weight average molecular weight (M _n, M _w), the quotient M _w/M _n the so-called dispersity (Đ), formerly known as polydispersity index (PD), indicating the measure of broadness of MW distribution, can be estimated. The presence of sulfate groups plays only a minor role here. Thus, estimation of the MW by GPC and the determination of the sulfate content by means of elemental analysis is a common approach for characterizing sulfated GAG (Schiller et al. 2010).

Fourier transform-infrared spectroscopy (FT-IR)

Although FT-IR is a very sensitive method for structure elucidation, it is rather less common for the characterisation of native GAG due to their polyfunctional character leading to a variety of peaks, which are often difficult to interpret. However, it is justified in the functionalization of GAG with specific, e.g. unsaturated moieties, such as (meth)acrylates, resulting in FT-IR spectra with characteristic absorption bands for these functional groups in defined regions of the spectrum. Compared to other methods of structural analysis (NMR, MS), FT-IR is a fast and simple method to qualitatively prove the success of a dedicated chemical modification (Becher et al. 2013).

NMR techniques

Since applications of solid-state NMR for GAG are rather limited, we will focus here on classical solution-state NMR. The molecular weight of the GAG has a significant impact on the spectral quality and particularly the ¹H-NMR resonances are broadened beyond the detection limit. This makes the analyte hardly detectable but simplifies the detection of “small” impurities such as alcohols, which were used as precipitating agents.

Nevertheless, GAG polysaccharides are generally characterized by ¹³C- not by ¹H-NMR (Schnabelrauch et al. 2013). Native GAG, particularly HA and CS, can be easily differentiated by ¹³C-NMR (Bociek et al. 1980). As the HA disaccharide repeating unit contains 14 different carbon atoms, 14 different ¹³C-NMR resonances can be observed and assigned (Schnabelrauch et al. 2013). Unfortunately, the introduction of additional sulfate residues (Figure 5) reduces the frequency dispersion making assignments often difficult – even at high field strengths. A powerful technique to determine whether there is an –OH group or a –O–SO₃H at a dedicated position relies on H/D exchange. The spectrum is acquired in H₂O and subsequently in D₂O. If there is no change in the chemical shift between both there is a sulfate residue, otherwise there is a free hydroxyl group (Bush et al. 1987). In Figure 5, the C-6 resonance of GlcNAc vanishes with sulfation, indicating that sulfation is preferred at this position.

Figure 5:

Comparative ¹³C-NMR studies on the regioselectivity of HA sulfation. Arrows show the effect of low-field shift of sulfated C-6 (sHA, DS_S = 1.0) and the signal clearance in the C-2/C-3 region after high degree of sulfation (DS_S = 3.0). Reprinted with permission from Becher et al. (2012). © 2012 American Chemical Society.

Mass spectrometry

There are many problems towards MS analysis of GAG (Pepi et al. 2021). The most important ones can be summarized as follows:

1. It is very difficult to transfer a polymeric compound into the gas phase without pronounced decomposition. This particularly applies for (highly polar) sugars.

2. The investigation of sulfated GAG is even more difficult because the introduction of sulfate residues increases the polarity. Additionally, there is a high probability to fragment the sulfate residues in the gas phase (Lemmnitzer et al. 2021).

3. All polysaccharides are characterized by a typical mass distribution. This reduces the sensitivity to detect a dedicated molecule.

4. Even if such a high MW carbohydrate would be detectable by MS, the corresponding peak would be significantly broadened. The extent of sulfation also plays a significant role: dextran with about 75 kDa could be successfully detected while heparin was only detectable at <6 kDa.

Thus, characterizing high MW GAG by MS is not feasible and previous degradation is unequivocally necessary to obtain detailed structural information. This will be outlined in another chapter of this special issue (Rademann et al. Sulfated HA oligosaccharides).

Collagen/GAG-based coatings

PLGA scaffolds (∅ 12 mm, height: 2 mm) with mono- and bimodal pore distribution revealed a homogenous aECM coating outside and inside the scaffold as well as a comparable amount of coll type I (290–320 μg) and sHA3 (about 170 μg) immobilized, as determined by colorimetric assays (Wojak-Ćwik et al. 2019). Thus, the larger surface area for scaffolds with bimodal pores did not significantly increase the coated aECM amount.

In contrast, TriLA scaffold (∅ 5 mm, height: 3 mm) with a higher average pore size and a broader distribution of individual pore sizes displayed higher amounts of immobilized coll (+44%) and sHA3 (+25%) compared to scaffolds with lower porosity (Krieghoff et al. 2019). The total amount of coll adsorbed per scaffolds was quantified between 10 and 25 μg/scaffold after incubation at 37 °C for 1 h in PBS. The coating efficiency for coll/sHA3 was found to be reduced compared to the coll coating only. However, both coatings were found to be stable at 4 °C for 7 days with a marginal loss of coll and sHA3 indicating storability to a certain extent (Figure 6).

Figure 6:

Characterization of aECM coatings on TriLA scaffolds. Qualitative analysis of coll and sHA3 on low (LoPo) and high porosity (HiPo) scaffolds as detected after 1 h at 37 °C and after 7 days at 4 °C using Sirius red and Toluidine blue, respectively. Scale bar: 5 mm. Reprinted from Krieghoff et al. (2019) with permission from Biomaterials Research.

The amount of collagen adsorbed to PCL scaffolds (∅ 5 mm) by immersion in fibrillogenesis solution containing various GAG derivatives was again found to be highest for coll only and decreased to about 50% with the inclusion of CS or sHA3 (Förster et al. 2020; Neuber et al. 2019). The total amount of coll adsorbed was between 16 and 44 μg/scaffold and GAG between 2.6 and 3.5 μg/scaffold.

Acrylated hyaluronan/collagen-based hydrogels

Photocrosslinking of HA-acrylate (HA-AC) with or without covalently linked acrylated, sulfated GAG (sGAG-AC) and non-covalently embedded fibrillar coll type I led to dimensionally stable hydrogels (Rother et al. 2017, 2021; Thönes et al. 2019). The coll fibrils were included without any further crosslinking to preserve its native structure and properties. After lyophilization, the hydrogels showed porous structures with pore diameters of >100 µm as revealed by scanning electron microscopy (SEM) analysis (Rother et al. 2017). HA/coll gels with and without sGAG-AC displayed coll fibrils with periodic cross-striation, while surfaces of gels without coll appeared smooth (Figure 7A–D).

Figure 7:

Selected aspects of HA-AC hydrogel characterization. A–D: SEM images of hydrogel morphology with cross-sections of HA-AC hydrogels without (A, C) or with coll (B, D). Scale bars: 400 μm upper panel, 2 μm lower panel; arrows highlight the banding pattern of fibrils. E: Lysozyme release from different HA-AC/coll hydrogels relative to the initial amount of gel-bound enzyme during incubation in 2% BSA/PBS at 37 °C. Two-way ANOVA: ^# p < 0.05; ^### p < 0.001 versus HA-AC/coll; *p < 0.05; **p < 0.01; ***p < 0.001 versus respective gel composition. Reprinted from Rother et al. (2017) with permission from Macromolecular Bioscience.

The swelling properties of these gels were determined gravimetrically with the swelling ratio and the water content before and after lyophilization as well as by measuring the loss of volume with a digital measuring slide (Rother et al. 2017). The gel mass increases due to water binding in comparison to the initial weight after freeze-drying (swelling ratio), but was significantly diminished in the presence of coll type I (HA-AC/coll). This might result from space exclusion of water molecules by embedded coll fibrils. It increased in the presence of sGAG-AC reaching the swelling ratio of HA-AC without coll type I suggesting compensation for the coll effect due to their higher water binding capacity. Also, including coll fibrils led to a significantly decreased water content even though values were rather similar reaching 97–99%. Likewise, the addition of coll led to significant reduction in the loss of volume after lyophilization. The elastic moduli were found to be in the lower kPa range with embedded coll reducing the elasticity significantly, while being only marginally affected by sGAG-AC included. Here, embedded coll fibrils might sterically hinder the crosslinking of acrylated groups resulting in a decreased number of crosslinks per gel volume.

The gels demonstrated a high stability regarding the cross-linked HA-AC and sGAG-AC content in PBS over 8 days at 37 °C. While there was almost no release of HA-AC detectable during this time, >90% of the originally used amount of sGAG-AC remained within the gels. In contrast, there was an enhanced loss of the non-covalently incorporated coll fibrils (30–50% after 8 days) in particular for sGAG-AC containing gels.

Photocrosslinked HA-AC hydrogel networks with non-covalently included aECM per se or in microgels were demonstrated to be rather stable in HA-AC and coll composition with about 93–94% of HA-AC and 70–80% of the initial coll contents remaining over 8 d. This is irrespective of the hydrogel composition (Rother et al. 2019). The reduced loss of coll in comparison to the study of Rother et al. (2017) might be explained by the higher HA-AC concentration used (5 vs. 1%), leading to a tighter network structure hindering coll diffusion. Further, when sHA3 was embedded via aECM-containing microfluidic derived microgels, a significantly enhanced retention of sHA3 was observed.

Collagen/GAG-based coatings

Consistent with earlier reports, the presence of coll and sHA3 significantly increased hMSC numbers and the activity of alkaline phosphatase (ALP), an early marker of early osteogenic differentiation, on PLGA scaffolds compared to uncoated ones, irrespective of the pore distribution (Wojak-Ćwik et al. 2019). However, enhanced mineralization and mRNA expression of osteogenic markers was evident for coated scaffolds with bimodal pore distribution suggesting that the latter is more beneficial for achieving osteogenic differentiation. This was attributed to a higher surface area, improved medium and signaling molecule exchange by the additional pores.

Likewise, an increased ALP activity and mineralization was found on coll/sHA3-coated TriLA scaffolds with more pronounced effects for scaffolds with higher porosity, ascribed to the higher coating efficiency achieved (Krieghoff et al. 2019). The underlying mechanism of aECM action, however, has not been completely clarified so far, but differences in cell adhesion and actin cytoskeleton signaling might play a role (Kliemt et al. 2013).

Critical size femur defects in rats treated with coll/sGAG-coated PCL-scaffold displayed increased coll synthesis and initial mineralization of the organic matrix as revealed by micro-computed tomography, biomechanical testing, NMR and histology (Förster et al. 2020). The highest de novo coll production was observed in the case of coll/CS, while coll/sHA3 induced new bone volume formation similar to autologous bone (positive control) 12 weeks after surgery, suggesting that coll coatings with sGAG strongly contribute to bone regeneration.

Another promising biomaterial strategy to achieve an osteogenic surface is by cross-copolymerized three-armed macromer-based polymer films covalently decorated with sHA3 (Gronbach et al. 2020). The surfaces efficiently scavenged Dickkopf-1, a strong inhibitor of the Wnt signaling pathway produced by SaOS-2 cells and primary hMSC, resulting in increased osteogenic differentiation.

Acrylated hyaluronan/collagen-based hydrogels

The enzymatic degradation profiles of the photocrosslinked HA-AC gels with or without covalently linked sGAG-AC and non-covalently embedded fibrillar coll type I were assessed using supraphysiological activities of hyaluronidase (Rother et al. 2017). Including coll led to a marked reduction of hydrogel degradation. While coll containing gels completely decomposed within 24–48 h at 37 °C, pure HA-AC gels were degraded after 3–6 h. There was no additional reducing effect for sGAG-AC-containing gels compared to HA-AC/coll. This was in contrast to a previous study by Feng et al. (2017) but likely related to the different degrees of sulfation used. The reduced degradation rate due to embedded coll fibrils might be favorable for improving clinical translation of HA hydrogels.

The GAG composition and sulfation was varied in the discussed hydrogels to adjust the biochemical properties for modulation and in-depth analyses of GAG–mediator and GAG–cell interactions. The presence of CS-AC and sHA1-AC significantly increased the amount of bound lysozyme, used as a model protein, within hydrogels (Rother et al. 2017). Additionally, sGAG-AC-containing gels significantly retarded lysozyme release when hydrogels were incubated in PBS at 37 °C for 8 days with the highest release within the first 4 h (Figure 7E). Applying HA-AC/coll and sHA1-AC-modified hydrogels in binding and release of heparin-binding epidermal growth factor-like growth factor (HB-EGF) for tuning skin cell responses, then likewise revealed that including sHA1-AC led to a higher HB-EGF binding of gels (36.7% ± 12.7 vs. 28.8% ± 16.1%) (Thönes et al. 2019). More importantly, the release was significantly retarded over 3 days in cell culture medium by incorporating sHA1-AC with about 1 ng HB-EGF released per 24 h.

Further, slightly higher amounts of vascular endothelial growth factor (VEGF) were bound by CS-AC-, sHA1-AC- and sHA1Δ6S-AC-containing gels compared to pure HA-AC/coll (Rother et al. 2021). However, even more striking, the addition of sGAG-AC decreased the VEGF release depending on the sGAG-AC type in the following order: HA-AC/sHA1-AC/coll < HA-AC/sHA1Δ6s-AC/coll < HA-AC/CS-AC/coll ≈ HA-AC/coll. This continuously slower release due to the presence of sGAG-AC for all three proteins suggest GAG-specific interactions instead of non-specific protein adsorption. These studies further demonstrate that sGAG-functionalized hydrogels are able to tune the interaction profiles with proteins and have a high potential for serving as protein carriers or scavenging systems in vivo (Rother et al. 2017, 2021; Thönes et al. 2019). They are also in line with a study by Feng et al. (2017) showing an improved TGF-β sequestration and extended availability in methacrylated HA hydrogels when crosslinked with methacrylated sHA, promoting chondrogenesis of encapsulated hMSC.

Analyzing the interaction of photocrosslinked HA-AC hydrogels with non-covalently included aECM per se or in microgels with transforming growth factor-β1 (TGF-β1) demonstrated that gels containing sHA3 bound significantly higher amounts of TGF-β1 in comparison to those without sHA3 (Rother et al. 2019). Importantly, the type of sHA3 integration influenced the retention of TGF-β1 within these gels with the aECM incorporation via microgels significantly decreasing growth factor release. This property could be beneficial for improving skin repair by re-balancing dysregulated TGF-β1-induced cellular responses.

While all investigated gel compositions of HA-AC gels with covalently linked sGAG-AC and non-covalently embedded fibrillar coll type I enabled cell migration and 3D cell expansion, only those with sHA-AC strongly promoted endothelial cell growth in vitro (Rother et al. 2017). Here, again differences in cell adhesion and actin cytoskeleton signaling might play a role (Kliemt et al. 2013). Further, it was found, that the sGAG-AC type included in HA-AC hydrogels strongly affects VEGF-related endothelial cell growth with the highest cell numbers on sHA-AC-containing gels loaded with VEGF (Rother et al. 2021). In addition, VEGF-loaded hydrogels containing CS-AC and sHA1Δ6s-AC but not sHA1-AC displayed elongated cell structures indicating sprouting. Increasing proliferation and promoting sprouting may result into enhanced angiogenesis during wound healing restoring the regeneration capacities of large-size vascularized tissue defects.

Further, HB-EGF released from HA-AC/coll gels and those modified with sHA1-AC stimulated the migration of a human keratinocyte cell line for at least 3 days and induced hepatocyte growth factor (HGF) expression in human dermal fibroblast (Thönes et al. 2019). Importantly, it was demonstrated, that HB-EGF released from sHA1-AC-containing hydrogels was more efficient in supporting wound closure in a porcine skin organ culture model than solute HB-EGF directly applied to the wound. In summary, these findings demonstrate that acrylated and non-acrylated sGAG included in HA-AC/coll-based hydrogels bind and release biologically active growth factors in a distinct and GAG-dependent manner. By increasing growth factor effectivity on target cells, they indicate great promise as mediator releasing wound dressings or biomaterials for promoting wound healing of damaged, vascularized tissues. Still, further in vivo studies are necessary to validate these promising features.