Injectable biodegradable gelatin-methacrylate/β‐tricalcium phosphate composite for the repair of bone defects
Graphical abstract
Introduction
Teeth and periodontal tissues weaken over time and are difficult to recover once they are damaged. Therefore, as age increases, the number of patients with periodontal disease continues to increase. Periodontal disease is one of the most common diseases suffered by about 90% of the adult population over the age of 70. In addition, when periodontitis persists, alveolar bone absorption and loss of teeth may occur due to inflammation [1], [2], [3]. Alveolar bone absorptions and defects are also caused by various incidents such as trauma, genetic factors, cancer, and tooth decay. In order to restore functional and aesthetic characteristics of the defected area in a clinical setting, the lost bone tissue is regenerated using alveolar bone graft, guided tissue/bone regeneration (GTR/GBR) techniques, and then treatment is carried out through implant placement [4].
Currently, the most commonly used method for the repair of damaged alveolar bone is to transplant bone-shaped material directly to the defect site. The bone graft materials (BGM) are classified as autologous bone, allogeneic bone, and synthetic bone, depending on the raw materials. In the past, use of autologous bone is regarded as the “gold standard” due to its high bone formation/induction potential. However, it requires a secondary surgery to collect the bone from another location in the body and increases the possibility of damage in the donor site. In clinical practice, BGM are often provided as a powder and are usually mixed with blood or saline to maintain shape during the procedure. This procedure is easily applied for treatment of small defects. However, for larger defects, it is difficult to shape and form the reconstituted BGM due to weak adhesion by blood or saline solution. In this case, a mesh-type auxiliary device, such as titanium mesh, can be used to maintain the reconstructed shape. However, it requires a second operation to remove the mesh after the bone regeneration is completed, thereby raising concerns of infection and prolonging the recovery period (See Fig. 1).
In order to solve these difficulties, the use of synthetic bone has been steadily increasing [5]. The use of synthetic bone materials, such as hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) as the main materials, has been developed and much research has been carried out to improve the functionality of synthetic bone [6], [7]. Leupold et al. and Mojgan et al. established that HA, or natural polymer-HA composites, have good bone regenerative capability in bone defect animal models. However, in both studies, HA remained in the regenerated region and could not be completely replaced with new bone [8], [9]. HA is a major constituent of bone and has high biocompatibility and high strength, which plays a role in complementing the strength of β-TCP. However, since biodegradability is delayed, the prolonged residence time in the body can interfere with bone remodeling [10]. In contrast, β-TCP is readily degradable and induces osteoconduction through internal micropores to form new bone. It is known to be completely degraded and does not remain in the tissue after bone formation, thus allowing for complete bone regeneration [11].
Gelatin is a kind of induction protein derived from collagen, a natural protein that constitutes animal skin, tendon, and cartilage, and has many advantages such as excellent biodegradability, biocompatibility and non-toxicity [12], [13], [14], [15]. Gelatin/methacrylic anhydride (GelMA), which has a methacrylate group added to the amine group present in the gelatin, can be made into photo-crosslinked hydrogels. The long-term biocompatibility of GelMA hydrogels has been demonstrated through many studies [16]. In addition, GelMA hydrogels have been extensively applied to tissue regeneration, through cell culture and drug delivery in the field of tissue engineering. Here it has advantages due to its similarities with extracellular matrix [17], [18]. However, GelMA hydrogels have lower mechanical strength than other hydrogels, such as alginate and polyethylene glycol diacrylate [19], [20]. Therefore, it is necessary to improve the mechanical strength by adding other substances in order to apply GelMA hydrogels to bone tissue regeneration. For these reasons, we designed a composite with GelMA hydrogel incorporating β-TCP (GelMA-B). GelMA hydrogel can maintain the reconstructed shape itself during the healing period by applying a bonding force to the β-TCP (Fig. 1).
Recently, a hydrogel system containing bio-ceramic has been studied for effective bone regeneration. Iviglia et al reported that pectin/chitosan hydrogel filled with hydroxyapatite/β-TCP demonstrated a pro-osteogenic response. Additionally, Chun et al studied the bone tissue regeneration applicability of CaP incorporated in a hydrogel system [21], [22]. However, further studies to confirm the bone regeneration effect of these hydrogel systems directly are required.
In this study, we analyzed the chemical and physical properties of the prepared GelMA and GelMA-B. In addition, their biocompatibility and bone regeneration effect were confirmed through in vitro and vivo experiments on human adipose-derived stem cells (hASCs) and in a rat model.
Section snippets
Materials
Gelatin (type A) and methacrylic anhydride (MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The photo-initiator, 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propanone (Irgarcure 2959), was purchased from Ciba Specialty Chemicals (Basel, Switzerland). The BGM, beta-tricalcium phosphate (β-TCP, Cerasorb® M), was purchased from Curasan AG (Kleinostheim, Germany). Spectra/Por 4 dialysis tubing (12–14 kD MWCO, 45 mm flat-width) was purchased from Spectrum Laboratories Inc (CA,
Preparation and characterization of GelMA hydrogels and GelMA-B
To confirm the binding of MA to the amine groups of gelatin, synthesized GelMA monomer was evaluated by 1H NMR measurement in D2O (Fig. 2A). In the 1H NMR spectra of GelMA monomer, new peaks appeared at 5.6, 5.3 and 1.8 ppm. The gelatin and GelMA monomer amide II (1530 cm−1) and amide III (1230 cm−1) bands appeared at the same wavenumbers in the FT-IR analysis (Fig. 2B). However, the GelMA of amide I (1640 cm−1) showed a peak at slightly higher wavenumber than gelatin (1620 cm−1). In addition,
Discussion
The synthesis of GelMA monomer, a photo-crosslinkable hydrogel applied to β-TCP, was confirmed by confirming the presence of methacrylamide or methacrylate bound to the gelatin backbone through 1H NMR analysis. In Fig. 2A, the peak of the acrylic protons (a) and the hydroxyl lysine group (b) in the lysine group of methacrylamide appeared at 5.6 and 5.3 ppm and the methyl protons (c) peak was observed at 1.8 ppm [30]. Therefore, methacrylate/methacrylamide groups had been successfully grafted
Conclusions
We prepared a GelMA/BGM composite for bone regeneration. This gel could maintain the reconstructed shape at the bone defect site without additional equipment. The mechanical properties of the produced materials were stronger than GelMA hydrogel or BGM used alone. This was verified by viscoelastic measurements. The biocompatibility and osteo-differentiation effect of GelMA-BH were demonstrated using hASCs and measuring cell-viability and ALP activity, respectively. Moreover, GelMA-BH had
Acknowledgements
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2017M3A9E4048170).
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These authors contributed equally to this work.