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. Author manuscript; available in PMC: 2023 Dec 5.
Published in final edited form as: Mol Pharm. 2022 Jun 8;19(12):4565–4575. doi: 10.1021/acs.molpharmaceut.2c00141

High Molecular Weight poly (Glutamic acid) to Improve BMP2-induced Osteogenic Differentiation

Jue Hu a,b, Zhuozhi Wang a,b, Jacob M Miszuk a,b, Erliang Zeng b, Hongli Sun a,b,*
PMCID: PMC9729371  NIHMSID: NIHMS1820709  PMID: 35675584

Abstract

FDA-approved bone morphogenetic protein 2 (BMP2) has serious side effects due to the super high dose requirement. Heparin is one of the most well-studied sulfated polymers to stabilize BMP2 and improve its functionality. However, the clinical use of heparin is questionable because of its undesired anticoagulant activity. Recent study suggests that poly (glutamic acid) (pGlu) has the potential to improve BMP2 bioactivity with less safety concerns, however the knowledge on pGlu’s contribution remains largely unknown. Therefore, we aimed to study the role of pGlu in BMP2-induced osteogenesis and its potential application in bone tissue engineering. Our data, for the first time, indicated that both low (L-pGlu) and high molecular weight pGlu (H-pGlu) were able to significantly improve BMP2-induced early osteoblastic differentiation marker (ALP) in MC3T3-E1 pre-osteoblasts. Importantly, the matrix mineralization was more rapidly enhanced by H-pGlu compared to L-pGlu. Additionally, our data indicated that only alpha H-pGlu (α-H-pGlu) could significantly improve BMP2’s activity while gamma H-pGlu (γ-H-pGlu) failed to do so. Moreover, both gene expression and mineralization data demonstrated that α-H-pGlu enabled single-dose of BMP2 inducing high level of osteoblastic differentiation without multiple-dose of BMP2. To study the potential application of pGlu in tissue engineering, we incorporated the H-pGlu+BMP2 nanocomplexes into the collagen hydrogel with significantly elevated osteoblastic differentiation. Furthermore, H-pGlu-coated 3D porous gelatin and chitosan scaffolds significantly enhanced osteogenic differentiation through enabling sustained release of BMP2. Thus, our findings suggest H-pGlu is a promising new alternative with great potential for bone tissue engineering applications.

Keywords: Poly (glutamic acid), Bone morphogenetic protein-2, Drug release, Osteogenic differentiation, 3D porous scaffold, Bone tissue engineering

1. Introduction

Autologous bone grafting is the gold standard for treatment of large bone defects while it is significantly limited by availability and the risk of donor site morbidity.1, 2 Various strategies to stimulate bone regeneration have been investigated,3–5 among which, biomaterial-mediated growth factor delivery is a promising alternative to autologous bone grafts.6

FDA-approved bone morphogenetic protein-2 (BMP2), a member of the transforming growth factor-beta (TGF-β) superfamily,7 has been widely used in the treatment of spinal fusion,8 large bone defects,9 and oral rehabilitation.10 Since BMP2 is easily denatured under physiological conditions, extremely high doses of BMP2 are required to maintain its bioactivity for sufficient bone regeneration.11, 12 However, super high dose of BMP2 causes serious adverse effects, including postoperative inflammation reaction,13 neoplasms,14 and abnormal bone formation.15 This has urged us to develop innovative drug delivery techniques to address these challenges of BMP2 application.16–18

Sulfated polymer heparin has received extensive attention as it can increase the efficacy of BMP2 by protecting its bioactivity.19–22 The negatively charged heparin and the positively charged BMP2 could form a polyelectrolyte complex,23, 24 stabilizing BMP2 from degradation and enhancing its osteo-inductive capability. However, the clinical applications of heparin were limited because of its strong anticoagulant activity.25, 26

Poly (glutamic acid) (pGlu) is a highly negatively charged amino acid biopolymer that recently attracted attention with promise in biomedical applications by virtue of its biological safety, non-immunogenicity, and biodegradability.27–30 Although several studies have tested the use of pGlu in bone tissue engineering, most of recent work focused on the application of gamma type of pGlu (γ-pGlu) as scaffolds and hydrogels.31–34 Interestingly, one recent study found pGlu shows some promise to interact with BMP2 and improve its activity,34 indicating the possibility of using pGlu as an alternative to heparin. However, only low molecular weight pGlu was studied in this research. pGlu is classified into poly (alpha-L-glutamic acid) (α-L-pGlu) and poly (gamma-glutamic acid) (γ-pGlu), according to the chemical bonds between the amino group and the carboxyl group.27 The molecular weight, dose, and chemical structure may have significant impacts on the interactions of pGlu with BMP2 and the subsequent functions which have not been studied so far. This knowledge gap will significantly limit the potential applications of pGlu in bone tissue engineering.

In this study, for the first time, we reported that only α-pGlu can effectively improve the bioactivity of BMP2 while γ-pGlu has bare contributions. Moreover, higher molecular weight alpha type pGlu (α-H-pGlu) has better ability to improve the BMP2-induced osteogenic differentiation than the lower molecular weight pGlu (L-pGlu). The H-pGlu/BMP2 nanocomplexes can be easily loaded into a hydrogel to improve BMP2-induced osteogenic differentiation. We further demonstrated that H-pGlu can enable sustained release of BMP2 from three-dimensional (3D) scaffolds through facile coating which revealed the promising application potential of H-pGlu in bone tissue engineering.

2. Experimental

2.1. Materials

Low/high molecular weight alpha type poly (α-L-glutamic acid) (L-pGlu; molecular weight:1500 to 5500; H-pGlu; molecular weight: 50 k-100 k) and high molecular weight gamma type poly (γ-L-glutamic acid) (γ-H-pGlu; molecular weight ≥ 750 k), Vanillin, ascorbic acid, β-Glycerophosphate salt, and 100% ethanol were purchased from Sigma (St. Louis MO, USA). Chitosan (85% deacetylated) was purchased from INDOFINE chemical company, Inc (Hillsborough, NJ, USA). Bioglass (BG, 45% SiO2 + 24.5% Na2O + 24.5% CaO + 6% P2O5, >98%, 10μm) was bought from MO-SCI ONLINE (North Rolla, MO, USA). Bovine Serum Albumin (BSA) was purchased from Fisher Scientific (New Jersey, USA). Type I Collagen Solution, 4 mg/ml (Rat Tail) was purchased from Advanced BioMatrix (Carlsbad, CA, USA). Recombinant human BMP2 (rhBMP2) and BMP2 Mini ABTS ELISA Development Kit were bought from PeproTech (Rocky Hill, NJ, USA). Other chemical reagents were of analytical grade.

2.2. Cell culture

Minimum Essential Medium Alpha (1X) (α-MEM, Gibco, Waltham, MA), supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin sulfate, and 100 U/mL penicillin was used as the normal Growth Medium. Mouse calvarial-derived pre-osteoblasts (MC3T3-E1, from ATCC) were cultured in Corning® T-75 flasks under a humidified atmosphere with 5% CO2 at 37°C. Growth medium was changed twice per week. MC3T3-E1 cells were passaged when they reached around 90% confluence. Passages between 8 and 10 were used for this research. To induce MC3T3-E1 cells osteoblastic differentiation, cells were cultured in an osteoconductive medium (growth medium containing 50 μg/mL L-ascorbic acid and 2.5 mM β-glycerophosphate salt).

2.3. Alkaline phosphatase (ALP) activity

1×104 MC3T3-E1 cells were seeded into a 24-well plate with growth medium and incubated at 37°C containing 5% CO2. After overnight incubation, the growth medium was replaced with an osteoconductive medium. After 7 days cell culture, the ALP activity was quantified and qualified with an EnzoLyte pNPP Alkaline Phosphatase Assay Kit (AnaSpec, San Jose, CA, USA), following the product manual. In brief, samples were rinsed with DPBS and lysed for 5 min at 25°C. Collected lysate was transferred into a centrifuge tube, incubated on ice for 10 min under mild shaking, and centrifuged for 10 min under 2500×g at 4°C. The supernatant/standard solution (50 μL) was mixed with p-nitrophenyl phosphate (25°C) and incubated at 37°C for 30 min. ALP activity was then tested at 405 nm and normalized against total protein content, which was measured by a BCA kit (Thermo Scientific™, Waltham, MA, USA) based on the user instructions. In short, 25μL collected supernatant (same from the former ALP activity experiment)/standard solution was homogeneously mixed with 200 μL of BCA working solution, incubated at 37°C for 30 min, and measured at 562 nm.

2.4. Characterization of the poly (glutamic acid)/BMP2 complexes

Dynamic Light Scattering (DLS) was used to test the size distribution of varied kinds of pGlu, and their complexes. Both BMP2 and pGlu were firstly dissolved in 10 mM sodium phosphate buffer, separately. Right before the test, BMP2 and the pGlu solutions were mixed at an equal volume ratio to form the polyelectrolyte complexes. DLS measurements were performed on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).

2.5. Alizarin red S (ARS) staining and quantification

ARS staining was used to detect the calcium-rich deposits by cells according to our previous protocols35, 36 with a minor modification. Briefly, after two to three weeks of cell culture, cells were rinsed with DPBS and fixed in 70% ice-cold ETOH for at least 1 h, washed with distilled water and stained by 40 mM ARS solution (Sigma, pH to 4.2 by 1 M hydrochloride acid) for 10 min at 25°C under shaking. For the quantification of the ARS staining, 10% w/v cetylpyridinium chloride (CPC) (Sigma St. Louis MO, USA) was prepared and added 1 mL/well and incubated for 30 min to elute the stain. The eluted dye was measured at 550 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

2.6. Quantitative gene analysis

The osteogenic biomarkers including alkaline phosphate (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were analyzed on day 7 using real-time PCR. Quantitative gene expression analysis was carried out according to the product instructions. Briefly, total cellular RNA was extracted using a miRNeasy Mini Kit (Qiagen, Valencia, CA). The concentration of total RNA was measured using a NanoDrop™ One spectrophotometer (Thermo Scientific, Pittsburgh, PA). Equivalent amount of RNA was processed to generate a complementary strand of DNA (cDNA) using an iScript™ cDNA Synthesis Kit (Bio-Rad Hercules, CA). ALP, OCN, and BSP were expressed on a CFX Connect™ (Bio-Rad Hercules, CA) utilizing the iQ SYBR® Green Supermix Kit (Bio-Rad Hercules, CA). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and other gene primers were synthesized at the University of Iowa (Table S1).

2.7. Potential applications of H-pGlu in bone tissue engineering

2.7.1. Preparation of H-pGlu/BMP2 incorporated collagen hydrogel and ALP activity study

Collagen hydrogels were prepared according to the user manual. In brief, 9 parts of 4 mg/mL collagen solutions (loaded with either 200 ng BMP2 alone or H-pGlu/BMP2 complexes) were mixed with 1 part of neutralization solution. Collagen hydrogel was formed in a 37°C incubator for 1 hour. Then a total of 100 μL hydrogels were floated in the growth medium (GM) of MC3T3-E1 cells. GM and collagen alone (Col) were used as negative control, while BMP2 multiple times treatment (BMP2 ×2) was the positive control. Varied doses of H-pGlu (with a final concentration of 1, 2.5, and 5 mg/mL) were mixed with BMP2 in advance. ALP activity was tested on day 7.

2.7.2. Preparation of 3D gelatin and Chitosan-Vanillin-Bioglass (CVB) Scaffolds and ALP activity study

Gelatin 3D scaffolds (Gel) and Chitosan-Vanillin-Bioglass (CVB) scaffolds were prepared according to our previous publications.17, 36 For the preparation of 3D Gel scaffolds, 40°C 50% v/v ethanol/water mixed solvent was used to dissolve gelatin type B to make a 10% w/v uniform gelatin solution. The porogen (150 to 300 μm sized paraffin spheres, PS) was prepared in advance for Gel scaffolds. Then 10% w/v gelatin solution was poured into PS template and immediately transferred into a −80°C freezer for overnight phase separation. The frozen samples were then immersed into ethanol and 1, 4-dioxane for 24 h, respectively, followed by −80°C overnight, and then 48 h lyophilization. The Gelatin-PS composite scaffolds were cut into 5mm width and 1mm height discs by Premier Biopsy Punch (Plymouth Meeting, PA, USA) and soaked in hexane to remove PS. Residual hexane was exchanged by fresh cyclohexane every 2 h for 8 h. Finally, samples were lyophilized for 48 h and stored in a desiccator for future experiments. In case of CVB scaffolds, firstly, chitosan was dissolved in 1% (v/v) acetic acid to get a 4% (w/v) solution. Secondly, vanillin solution and BG solutions were mixed. Afterwards, 500 μl mixture was slowly added dropwise into 2 mL chitosan solution prepared above to induce gelation. The gelation time was around 5 to 7 min. Mass ratios of chitosan: vanillin: BG was kept at 32:16:2.5. The CVB scaffolds were prepared by freezing the CVB hydrogel at −80°C overnight followed by lyophilization for 48 hours.

Gel and CVB scaffolds were sterilized by 70% ETOH followed by 3 times wash by DPBS and soaked in DI water for further use. The sterilized scaffolds were divided to control group and H-pGlu coated group. For H-pGlu coated groups, the Gel and CVB scaffolds were dried by sterilized gauze and immersed in 5 mg/mL H-pGlu aqueous solution for 24 hours followed by rinsing in DI water and drying by gauze. An equivalent amount of BMP2 (200 ng in 4 μL) was dropped into both neat scaffolds and H-pGlu coated scaffolds and incubated for 30 min to ensure the full binding of BMP2 to respective scaffolds. Finally, treated scaffolds were then suspended in 500 μL cell culture medium without touching the cells at bottom (one scaffold per well). GM medium without scaffolds (GM), BMP2 multiple times treatment (BMP2 ×2, total BMP2 = 200 ng) and Gel/CVB alone were used as control groups. ALP activity was tested on day 7.

2.7.3. Cationic dye staining

Cationic dye staining was used to indicate the H-pGlu coating. Crystal violet can be dissociated into positively charged ions in aqueous solution. The anionic H-pGlu is stainable by cationic dyes. Therefore, neat 3D Gel/CVB or H-pGlu (5 mg/mL) coated Gel/CVB scaffolds were stained with 0.1% (w/v) crystal violet for 10 min and then thoroughly rinsed with distilled water for 10 min. All the samples were taken images by a Canon digital camera.

2.7.4. Characterization of pGlu loaded hydrogel and scaffolds

Surface microstructure and morphologies of lyophilized hydrogel and scaffolds were characterized by a Hitachi S-4800 Scanning Electron Microscopes. Samples were sputter coated with gold and imaged at an accelerating voltage of 5 kV.

2.8. RhBMP2 release from 3D scaffolds

The influence of H-pGlu for BMP2 release were studied on both 3D Gel and CVB scaffolds. The preparation of H-pGlu coated scaffolds was the same as in section 2.8.2. Then 200 μL 1x DPBS (with 0.1% bovine serum albumin) was used as release buffer and placed in a 37°C shaker. The BMP2 release supernatants were removed and replaced with fresh release buffer at specific time points. After 7 days of release, a human/murine/rat BMP-2 ELISA kit (Peprotech, USA) was used per our previous protocol17 to detect BMP2 release from the scaffolds.

2.9. Hydroxyapatite (HA) deposition ability of scaffolds with or without H-pGlu

For HA coating, 3D scaffolds (with a size of 5mm ×5mm ×2mm thickness) were immersed in a 10 times simulated body fluid (10× SBF) solution and then incubated at 37 °C in a closed Falcon tube for predetermined periods. The SBF solution was prepared according to the previously reported method.37 Firstly, 450 mL of DI water was measured. Secondly, 29.22 g NaCl, 0.19 g KCl, 1.38 g CaCl2, 0.58 g MgCl2∙6H2O, 0.28 g NaH2PO4∙2H2O, 0.42 g NaHCO3 were added to above DI water. After the final ingredient completely dissolves, the solution was then adjusted to 500 mL using DI water, and pH adjusted to 6.0. After a specific time (day 3 and day 7), the scaffolds were removed and washed three times with distilled water to remove unattached ions. After that, the scaffolds were lyophilized and characterized using the SEM for biomineralization capacity.

2.10. Statistical analysis

Three independent experiments at least, unless otherwise stated, were performed and data were presented as mean ± standard deviation. The means for three or more parallel groups were analyzed by one-way ANOVA with post hoc tests. *P < 0.05; **P < 0.01; ***P < 0.001 were identified as statistically significant between individual groups.

3. Results

3.1. Influence of molecular weight, dose, and chemical structure of pGlu on BMP2 activity

The chemical structures of glutamic acid monomer, poly (α-L-glutamic acid), and poly (γ-glutamic acid) are illustrated in Figure 1 (A) Possible interactions between BMP2 and pGlu polymers (B) and the schematic diagram (C) of the preparation of BMP2 loaded Gel/CVB+H-pGlu scaffolds are also included in Figure 1.

Figure 1.

Figure 1.

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Chemical structure of (A) glutamic acid monomer, poly (α-L-glutamic acid), and poly (γ-glutamic acid), (B) possible interaction between BMP2 and pGlu polymers, (C) schematic diagram of the preparation of BMP2 loaded Gel/CVB+H-pGlu scaffolds.

We firstly compared the effects of the molecular weight of pGlu on BMP2’s bioactivity. As our data indicated, both low and high molecule weight pGlu (both are α type) can significantly improve the ALP activity (early osteogenic marker, tested on day 7) and in a dose dependent manner (Figure 2A). However, H-pGlu more effectively improved the mineralization (mature osteogenic marker) of MC3T3-E1 cells compared to the L-pGlu (Figure 2B) at the same dosage (500 μg/mL). Please note, the significant mineralization appeared as soon as 2 weeks after the treatment with H-pGlu. Compared to the control group (growth medium, GM), H-pGlu significantly and dose-dependently reduced the proliferation of MC3T3-E1 cells although no noticeable impact on the cell morphology was observed (Figure S1). In addition to molecular weight, we found that α-H-pGlu was better than γ-H-pGlu in improving BMP-2-induced ALP activity (Figure 2D) at the same dosage (500 μg/mL). DLS was used to investigate the polyelectrolyte complex formation of BMP2 with variety of pGlu (Figure S2). The average sizes of the α-H-pGlu were larger than γ-H-pGlu and α-L-pGlu. And there was an increased tendency for pGlu/BMP2 complexes to have a higher average size than pGlu alone. Therefore, we concluded that α-H-pGlu at 500 μg/mL was the strongest treatment to improve BMP2’s bioactivity and would be used for the rest of the study.

Figure 2.

Figure 2.

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Effects of the molecular weight, dose, and chemical structure of pGlu on BMP2-induced osteogenic differentiation and 2-week rapid mineralization. (A) Influence of molecular weight and dose (50, 250, 500 μg/mL pGlu) on ALP activity, (B) Alizarin Red S staining on day 14, (C) CPC quantification of MC3T3-E1 cells after treating with L-pGlu/BMP2 and H-pGlu/BMP2 mix in osteoconductive (OC) medium for 14 days, (D) Influence of different chemical structure of pGlu (500 μg/mL). Data are expressed as mean ± SD, n = 3. (*P < 0.05, **P < 0.01 vs. control, ns=no significant difference, BMP2 ×2 and BMP2 ×4 mean that the group was treated by BMP2 at 100 ng/mL for 2 and 4 times, respectively).

3.2. Effects of H-pGlu on osteoblastic differentiation

Importantly, mineralization data at week 3 indicated that one dose of H-pGlu (H-pGlux1) could more significantly improve mineral deposition compared to single dose treatment of BMP2 (Figure 3, ***P < 0.001). The highest mineralization was found in the H-pGlu+BMP2 mix x1 group, which is consistent with our Alizarin Red S staining data on week 2 (Figure 2B&C). The quantitative gene expression of osteogenic markers ALP, OCN, and BSP was evaluated to further study the effect of the H-pGlu on BMP2-induced osteogenic differentiation on day 7. Only H-pGlu+BMP2×1 group revealed an upregulation tendency of ALP gene marker, while H-pGlux1 group had no difference compared to the OC control (Figure 4, left). Both BMP2×2 and H-pGlu+BMP2×1 groups demonstrated significantly higher mature osteogenic marker OCN expression compared to OC (Figure 4, middle, *P < 0.05). And there was an up-regulation trend for H-pGlu containing group, which was consistent with our mineralization data (Figure 3). The H-pGlux1 and H-pGlu+BMP2 ×1 groups demonstrated significantly higher BSP expression compared to the BMP2×1 group (Figure 4, right, **P < 0.01). Additionally, the BSP expression in H-pGlu+BMP2×1 group was even higher than BMP2×2 group (*P < 0.05), further indicating the capacity of H-pGlu for improving BMP2-induced osteogenic differentiation.

Figure 3.

Figure 3.

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Three weeks’ mineralization of MC3T3-E1 treated by H-pGlu alone or H-pGlu/BMP2. (A) 3 weeks of Alizarin Red S staining, (B) CPC quantification and (C) microscope images of MC3T3-E1 cells after treating with H-pGlu, BMP2, and H-pGlu/BMP2 mix in osteoconductive (OC) medium at low (scale bars = 200 μm, left panel,) and high (scale bars = 100 μm, right panel) magnifications. Data are expressed as mean ± SD, n = 3. (**P < 0.01, ***P < 0.001 vs. control, H-pGlu = 500 μg/mL, BMP2 ×1 and BMP2 ×5 means cells were treated by BMP2 at 100ng/mL for 1 and 5 times with medium change, respectively).

Figure 4.

Figure 4.

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Osteogenic marker gene expressions (ALP, OCN and BSP) were quantified by real-time PCR assay after 7 days’ culture. Results are presented as mean±SD (n = 3, *p < 0.05; **p < 0.01 vs. control, H-pGlu = 500 μg/mL).

3.3. Potential application of H-pGlu in bone tissue engineering

3.3.1. Application of H-pGlu in collagen hydrogel

Collagen hydrogel alone (Col), BMP2 incorporated collagen hydrogel (Col+BMP2), and H-pGlu+BMP2 loaded collagen hydrogels were floated in growth medium (GM) while MC3T3-E1 cells were seeded on the tissue culture plate in advance. Consistent with our ALP and mineralization data (Figure 2 and Figure 3), we also noted that H-pGlu could dose-dependently increase the BMP2-induced ALP activity (Figure 5A). A significant increase of ALP activity was found in Col + H-pGlu (5 mg/mL)/BMP2 group compared to that of the BMP2×2 group. Therefore, 5 mg/mL H-pGlu was chosen for further study on 3D scaffolds. SEM data showed that both micro-and macro-pore structure of collagen hydrogel was largely changed by incorporation of the 5 mg/mL H-pGlu in the hydrogel (Figure 5B&C).

Figure 5.

Figure 5.

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H-pGlu improves BMP2 activity in a collagen hydrogel. (A) ALP activity, (B-C) SEM images of lyophilized Collagen and Collagen/H-pGlu scaffolds at low (scale bars = 500 μm, left panel,) and high (scale bars = 10μm, right panel) magnifications (* = 5 mg/mL H-pGlu).

3.3.2. H-pGlu for BMP2 release from 3D porous scaffolds

To further study the potential application of H-pGlu in tissue engineering, we fabricated two 3D porous scaffolds, i.e., Gelatin (Gel) and CVB scaffolds. The cationic dye staining data showed that significantly more crystal violets were absorbed to the CVB scaffolds compared to Gel scaffolds after the H-pGlu coating (Figure 6A). This data indicated CVB scaffolds could bind more H-pGlu than Gel scaffolds. Neat Gel/CVB scaffolds, Gel/CVB+BMP2, and Gel/CVB+H-pGlu+BMP2 scaffolds were floated in the growth medium (GM). Consistent with the data from hydrogel (Figure 5A), H-pGlu coating could significantly improve the BMP2-induced ALP activity on both Gel and CVB scaffolds (Figure 6B and Figure S3). Notably, the Gel/CVB+H-pGlu+BMP2 groups showed dramatically higher ALP activity compared to that of the Gel/CVB+BMP2 groups. To understand the contribution of H-pGlu to BMP2-induced osteogenic differentiation, release profiles of BMP2 from both neat and H-pGlu coated Gel and CVB scaffolds were studied (Figure 6C). Standard curve of BMP2 can be found in supporting information (Figure S4). As expected, an initial burst release was observed from both of Gel+BMP2 and CVB+BMP2 groups. BMP2 was almost completely released within the first 3 days from both Gel and CVB+BMP2 groups. BMP2 released from H-pGlu coated scaffolds (Gel+H-pGlu+BMP2 and CVB+H-pGlu+BMP2) showed a lower burst release followed by a sustained release during the experimental period. It was noted more sustainable release of BMP2 was observed from the CVB scaffolds compared to the Gel even after the same coating with H-pGlu (Figure 6C). This could explain the highest ALP activity achieved from the same group (Figure 6A).

Figure 6.

Figure 6.

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H-pGlu improves BMP2 activity and sustained release of BMP2 from 3D Gel and CVB scaffolds. (A) Crystal violet staining photograph of Gel (upper panel) and CVB (lower panel) before (Left column) and after H-pGlu coating (Right column). (B) ALP activity (C) 7 days release properties from 3D scaffolds with or without H-pGlu coating. All the results were represented as means ± SD, n = 3. (* p < 0.05, ** p < 0.01, vs. controls. Green and red color in graph B represents the two compared curves, which are CVB+BMP2 and CVB+H-pGlu+BMP2, respectively, 5 mg/mL H-pGlu solution was used for coating scaffolds).

3.4. Effects of H-pGlu on hydroxyapatite deposition on Gel and CVB scaffolds

SEM data showed that both Gel and CVB scaffolds maintained their macro-porous structures after H-pGlu coating (Figure 7). The effects of H-pGlu coating on hydroxyapatite (HA) deposition capacity on both Gel and CVB scaffolds were tested (Figure 8 and Figure S5). The SEM data suggested that mineralization speed of H-pGlu coated Gel and CVB scaffolds were slower than that of the control groups at early stage (day 3, Figure 8A, Figure S5). However, much thicker, and more extensive HA deposition was observed on H-pGlu coated scaffolds after 7 days of soaking in 10 × SBF (Figure 8B, Figure S5), especially for the Gel scaffolds (Figure 8B).

Figure 7.

Figure 7.

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SEM images of Gel, Gel/H-pGlu, CVB, and CVB/H-pGlu scaffolds at low (scale bars = 300 μm, left panel,) and high (scale bars = 50 μm, right panel) magnifications (5 mg/mL H-pGlu solution was used for coating scaffolds).

Figure 8.

Figure 8.

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HA deposition on Gel and Gel/H-pGlu scaffolds after 3 (A) and 7 days (B) soaking in 10x SBF at low (scale bars = 200 μm, left panel,) and high (scale bars = 50 μm, right panel) magnifications (5 mg/mL H-pGlu solution was used for coating scaffolds).

4. Discussion

pGlu is composed of glutamic acids, which are the natural constituents of the human body.34, 38–41 Except for serving as biocompatible/bio-inert materials, e.g., hydrogels, the roles of these polycarboxylates in bone regeneration have been barely studied so far. Based on the highly negative charge, one recent study indicated pGlu can bind BMP2 and improve its efficacy in vitro.34 It was noted that other similar polycarboxylates, including poly (acrylic acid) (pAA), poly (methacrylic acid) (pMAA), and poly (aspartic acid) (pAsp) had very limited capacity to improve BMP2-induced osteogenic differentiation.34 This study strongly suggests other characteristics of pGlu, e.g., structure, molecule weight etc. contribute to its unique functions besides the charge.

α-pGlu and γ-pGlu are the two main types of pGlu with distinct structures from the different chemical bonds (Figure 1A). Chemically synthesized α-pGlu comprises only L-glutamic acid connected by amide linkages between the α-amino and α-carboxylic acid groups. Naturally produced γ-pGlu contains both D- and L- glutamic acid connected by amide linkages between the α-amino and γ-carboxylic acid groups.27, 42 Due to the production methods, α-pGlu usually has a more broad distribution of molecular weights than γ-pGlu.27 Both α-pGlu and γ-pGlu have been widely used as biopolymers because they are generally biodegradable, water-soluble, and non-toxic.27 Consistent to the previous report,34 our study indicated that α-L-pGlu at 500 μg/mL could significantly improve the activity of BMP2. However, we found α-H-pGlu was more effective in improving BMP2-induced osteogenic differentiation compared to α-L-pGlu. Moreover, γ-H-pGlu was less effective to improve BMP2’s bioactivity although it had even higher molecule weight. These differences from molecule weight and structure cannot be explained by the size of pGlu-BMP2 complex since no correlations between their function and size were found according to our DLS test (Figure S2). More mechanism study is needed in the future.

Notably, our long-term mineralization and gene expression data further indicated that one dose of H-pGlu alone and H-pGlu/BMP2 complex could improve osteoblast differentiation. Multiple doses of BMP2 were required to achieve sufficient differentiation without the addition of H-pGlu. Moreover, L-pGlu was not effective for long-term function (mineralization) although it worked for short-term (ALP). These interesting data suggested that the H-pGlu has a longer protection period for BMP2, which might be due to the suitable molecular weight. Since the high molecular weight pGlu has more tangles and more negatively charged carboxyl groups in the same volume,43 that might contribute to the electrostatic interaction and adsorption of more positively charged BMP2. Considering this capacity, the H-pGlu might capture and stabilize the endogenous BMP2 for stronger osteoblastic differentiation even without exogenous BMP2 presence.

Our studies demonstrated that H-pGlu had great potential applications in both hydrogel and 3D porous scaffolds (Figure 5~Figure 8). Importantly, the highest ALP activity in CVB+H-pGlu+BMP2 group highlighted that the positive surface charge of chitosan might contribute to more adsorption of negatively charged H-pGlu,39 which was demonstrated by crystal violet staining (Figure 6A). The higher density of H-pGlu on the surface of CVB+H-pGlu finally led to a stronger binding capacity with BMP2 compared to the Gel+H-pGlu+BMP2 group (gelatin scaffold). Our release data further confirmed that H-pGlu coated scaffolds not only reduced the burst release at the early stage but also provided controlled/sustained release of BMP2 (Figure 6C). Since pGlu has been found to have the excellent apatite-forming ability in SBF as it contains abundant carboxyl groups,31, 43 we tested the HA deposition capacity of H-pGlu coated Gel and CVB scaffolds (Figure 8, Figure S5). The slower heterogeneous nucleation of apatite deposition on scaffolds at the early stage might be due to the rapid interaction among surrounding Ca2+ and the dissociated H-pGlu from scaffolds. After several exchanges of fresh SBF, the coated H-pGlu on scaffolds accelerated the HA formation. The different HA deposition efficiency between Gel and CVB scaffolds might be due to the similar reason as above discussed that there were higher electric interactions between CVB and H-pGlu. Our result suggested that H-pGlu could accelerate HA deposition on scaffolds and had potential application in bone tissue engineering.

Based on our data above, we believe that highly negatively charged H-pGlu enhanced BMP2 osteoinductivity through the formation of a nanocomplex with the positively charged BMP2, protecting BMP2 from fast degradation and enabling controlled/sustained release of BMP2. However, H-pGlu was not capable of improving the efficacy of bFGF (Figure S6), though the bFGF family also has rich positively charged amino acid.44 In contrary, negatively charged heparin can increase the bioactivity of both BMP2 and bFGF through electrostatic interaction.44, 45 Our data strongly demonstrated that H-pGlu has a specific high affinity with BMP2 although the mechanism is still elusive.

5. Conclusions

In this study, for the first time, we reported that only α-pGlu can effectively improve the bioactivity of BMP2 while γ-pGlu has bare contributions. Moreover, higher molecular weight alpha type pGlu (α-H-pGlu) has better ability than the lower molecular weight pGlu (α-L-pGlu). The H-pGlu/BMP2 nanocomplexes can be easily loaded into hydrogel to improve BMP2-induced osteogenic differentiation. We further demonstrated that H-pGlu can enable sustained release of BMP2 from three-dimensional (3D) scaffolds through facile coating. Thus, our findings suggest H-pGlu is a promising alternative with great potential for bone tissue engineering applications.

Supplementary Material

Supplementary Materials

Acknowledgments

This work was supported by the startup funds from the Department of Oral and Maxillofacial Surgery at the University of Iowa, the National Institute of Dental & Craniofacial Research of the National Institutes of Health under Award Numbers R03DE027491, R01DE029159, and T90DE023520. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors also would like to thank Dr. Kevin G. Rice and the members of his lab for their support in DLS usage.

Footnotes

Supporting Information

The Supporting Information includes, Cell viability and morphology of MC3T3-E1 cells after treatment by H-pGlu; Size distributions of BMP2, pGlu, and pGlu+ BMP2; Absolute ALP activity of cells cultured on 3D Gel and CVB scaffolds; BMP2 ELISA Standard Curve; HA deposition on CVB and CVB/H-pGlu scaffolds; H-pGlu binding ability with bFGF; and Sequences of primers for real-time polymerase chain reaction.

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Supplementary Materials

Supplementary Materials
NIHMS1820709-supplement-Supplementary_Materials.docx (2MB, docx)

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