Evaluating Osteogenic Cell Differentiation Efficacy in the Presence of Polylactide Samples With Varied Compositions for Bone Grafting: In Vitro Study

Introduction

Bone reconstruction of jaws is the most complex area in modern dentistry and maxillofacial surgery. Bone defects can be caused by various factors, such as traumatic tooth extraction, injuries, tumor processes, infections, or birth abnormalities. Repairing these defects is a complex challenge requiring special techniques and materials.^1,2 When the dentofacial system is restored using dental implants, the quality and volume of the alveolar bone in the implantation area affects the position of the implant, its primary stability, and the repair of soft tissue shape.

After tooth loss, the alveolar bone is resorbed and atrophied, the width and height of the alveolar crest decrease, and the bone conditions become insufficient for implantation. Therefore, alveolar bone repair in the implantation area is crucial in dentistry.

According to current statistics, approximately 2.2 million bone augmentation operations are performed annually. Despite the large number of techniques, the problem of jaw atrophy remains relevant, and the number of such operations increases by 13% annually.² We do our best to decrease complications and risks and improve the performance quality of maxillofacial surgeons.

Currently, there are many world-known methods of bone augmentation, such as guided bone regeneration, augmentation with polytetrafluoroethylene membranes, collagen bioresorbable membranes, titanium meshes, augmentation with autoblocks, and others.^3,4 However, the main disadvantage of using ready-made titanium scaffolds is the intraoperative stage of the membrane adaptation by the individual dimensions of the patient defect. This significantly increases the operation time and can also affect the development of complications.⁵

Computer-aided design (CAD)/computer-aided manufacturing (CAM) technology can plan each case individually and create personalized titanium scaffolds for bone augmentation. Because they are personalized, the operation time is reduced, and the doctor does not need to adapt the product to the oral cavity. Moreover, considering the bone architecture and patient anatomical structure, the membrane is fixed, significantly reducing the risks.⁶ However, the placement of titanium membranes implies an additional surgical step: removing the membrane after bone maturation before implantation.^7,8

To improve the quality of maxillofacial surgery, materials with good mechanical properties and nontoxic and favorable biological and chemical characteristics are required to manufacture membranes for bone repair. Therefore, we decided to pay attention to individual but biodegradable membranes. Using bioabsorbable materials may be a new step in jawbone augmentation.^6,9 Indisputably, the most important advantage of these membranes is the withdrawal of 1 additional surgical stage: the scaffold removal before implantation.

Polylactide (PLA) is a biodegradable, biocompatible polymer widely used in medicine. It can manufacture various medical products and materials, such as sutures, implants, and even 3D-printed models.^9,10 One of the main advantages of PLA in medicine is its ability to resorb. This means that PLA can dissolve in the body and be eliminated naturally, excluding secondary surgery for material removal.

Polylactic-co-glycolic acid (PLGA) is also commonly used in medicine. Chemical crosslinking of lactic and glycolic acids significantly changes the properties of the polymer. For example, PLGA with a ratio of lactic and glycolic acid monomers (75:25) will have a smaller Young’s modulus relative to PLA and a higher degradation rate.^4,8,9 Depending on the monomer ratio and polymer molecular weight, PLGA resorption time can vary from several months to several years. Moreover, product thickness and shape; drugs or other additives; and environmental conditions, such as pH, temperature, and humidity, will have an influence.

The evaluation of biomaterials for guided bone regeneration necessitates comprehensive preclinical testing to confirm their safety, efficacy, and biodegradation profiles.^11–13 There are several types of preclinical tests:

• Hydrolytic degradation tests. These tests analyze the in vitro hydrolytic degradation behavior of membranes in phosphate buffer solution (PBS) at 37°C.

• Enzyme resistance tests. Samples are immersed in a 0.13% porcine trypsin solution and incubated at 37°C to assess their resistance to enzymatic activity.

• Bacterial collagenase resistance tests. Using a collagenase solution from Clostridium histolyticum, these tests simulate bacterial infection conditions to evaluate the degradation process.

• Cytotoxicity of materials. For example, the MTT assay evaluates the cytotoxicity on L-929 mouse fibroblast cells by quantifying the percentage of surviving cells and observing morphological changes.

• Mechanical tests. The static mechanical properties of the scaffolds are determined using a universal testing machine to evaluate tensile strength, Young’s modulus, and elongation at break.

• Scanning electron microscopy is used to assess the morphology of fibers at different stages of degradation in samples made from various materials. The form of polymer fibers, their adhesive structures, and other characteristics may vary.

Based on the literature analysis, scaffolds made of polylactide and its derivatives are a promising alternative to the application of titanium scaffolds in the maxillofacial area.¹⁴

The study aims to investigate the influence of different polymer compounds of individual scaffolds for bone repair on the proliferation and osteogenic differentiation processes in vitro.

Materials and Methods

Prototypes of membranes were prepared from pure PLA (Ingeo 4032D, NatureWorks LLC, USA), polylactide supplemented with 15% by weight of hydroxyapatite nanoparticles (HA, CAP 85UD, LLC “NPK” Polistom, Russia), PLGA (Lattice Services, 3D printing medical filaments) with monomer ratios 60:40 and 85:15.

Then, 10 × 10 × 0.5 mm plates were prepared by 3D printing using a BiZon Prusa i3 Steel PRO 3D printer (3DiY, Russia). The extruder temperature was 180°C, and the table temperature was 60°C.

Membranes were modeled in the ZBush program and then manufactured on a Prusa 3D printer by fused deposition modeling (FDM) printing followed by sterilization. This is a method of 3D model printing when plastic material (for example, PLA) melts and is applied in layers to create an object, and FDM printing is widely used in various fields, such as prototyping, manufacturing, medical technology, and design.^1,3

Samples were sterilized by gamma radiation (a method of radiation sterilization using particle flows capable of ionizing a substance). At the same time, the average dose during radiation sterilization is 25 kGy (2.5 Mrad).¹⁵ This value was chosen based on microbiological tests in accordance with GOST R ISO 11737-1 (Figure 1).

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Gingival fibroblasts were used in vitro to evaluate the proposed experimental samples from different polymeric materials on cell proliferation and osteogenic differentiation processes.

This study aimed to estimate the osteogenic differentiation and proliferation of cells in the presence of samples made of different polymeric materials. An in vitro protocol containing gingival fibroblasts was used. Gingival fibroblasts are multipotent cells of the connective gum tissue that play an essential role in maintaining the integrity and functionality of the gums, especially in the repair and regeneration of periodontal tissues.

Gingival fibroblasts at a concentration of 100 000 cells per well were seeded on culture plastic and cultured in low-glucose Dulbecco’s Modified Eagle Medium 1.0 (Gibco, USA) culture medium supplemented with 10% fetal bovine serum (HyClone, USA), 2 mM L-glutamine (Gibco, USA), 100 U/ml penicillin/streptomycin at 37°C, and 5% CO². After cell adhesion to the culture plastic, experimental samples of PLA composite samples 1–4 were placed in the wells (Figure 2).

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Osteogenic differentiation was induced by changing the culture medium with the appropriate one supplemented with the osteogenic differentiation factors, including 50 μg/ml ascorbic acid, 0.1 μM dexamethasone, and 10 mM β glycerophosphate. As a control, the cell cultures of gingival fibroblasts cultured in the presence of matrices in the control culture medium without osteogenic differentiation factors were used as well as cells cultured on plastic in the control and osteogenic media without composite materials. The cells were cultured for 20 days. The culture medium was changed 2 times a week. Provided experimental samples 1–4 were 3D opaque porous matrices that occupied most of the well volume. Therefore, we could not evaluate the proliferation and viability of cells under the matrices themselves during the cultivation process.

After the cultivation, the cell cultures were stained with alizarin red (Sigma, USA) to evaluate the influence of the experimental samples on the osteogenic differentiation of gingival fibroblasts that were cultured using matrices. Cells were flushed with PBS, fixed with 70% ethanol for 1 hour, washed with distilled water twice, and then incubated in alizarin red solution according to the manufacturer protocol for 20–30 minutes at room temperature. To visualize calcium phosphate deposits, the alizarin red stained cells were photographed using an inverted microscope (Leica, Germany). The spectrophotometric values were measured using a modified alizarin red extraction technique to analyze extracellular matrix calcification intensity quantitatively. A 10% acetic acid solution was added to the alizarin red stained cell cultures. They were then incubated for 10 minutes at room temperature; the color intensity was measured on a spectrophotometer at the required wavelength of 450 nm.

Results

During the culture process, we observed active proliferation of gingival fibroblasts at the matrix edges, indicating that the plates are not toxic and do not cause cell death (Figures 3 and 4).

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Although the cells actively proliferated in the presence of experimental samples, their proliferation was still lower than in the control cells, especially in sample 3. During the culture process, we observed that the culture medium changed color in the presence of sample 3, proving a change in the culture medium pH. We assume that this fact could negatively affect cell proliferation.

To fulfill the task and analyze the effectiveness of alizarin red staining, the experimental samples had to be taken out from the wells with cells.

Samples 1 and 2 were not changed though samples 3 and 4 were because they were in the culture medium for a long time, swelled, and changed their original shape; they became soft and plastic. As a result, when matrices were removed from the wells, they damaged a significant part of the cell monolayer, making further analysis more difficult. However, in undamaged areas, we could still assess the effect of experimental samples on the osteogenic differentiation of gingival fibroblasts cultured with plates.

As a result, it can be observed that gingival fibroblasts that were cultured under osteogenic conditions with samples 1–4 capable of osteogenic differentiation (Figures 5 and 6). But we did not find an increase in the induction of osteogenic differentiation in the presence of plates, regarding the control cells that were cultured in the medium with osteogenic differentiation factors without composite materials. However, when comparing samples 1 and 2 relative to each other, we found that the intensity of alizarin red staining in sample 2 was higher than in sample 1, indicating greater calcification of the extracellular matrix (Figure 5b). It was found that matrices supplemented with hydroxyapatite particles increase calcified extracellular matrix formation in gingival fibroblasts.

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However, when comparing samples 3 and 4 relative to each other, we found that the intensity of alizarin red staining in sample 4 was significantly higher than in sample 1, indicating greater calcification of the extracellular matrix (Figure 6b). We suggest that a change in the percentage of PLGA relative to PLA in the plate composition may cause an increase in calcified extracellular matrix formation in gingival fibroblasts.

Thus, when evaluating the effectiveness of osteogenic differentiation of gingival connective tissue cells on modified PLA and PLA/HA, PLGA 85/15, and PLGA 60/40 composite matrices, it was found that the cells were capable of proliferation and differentiation in the osteogenic direction in the presence of polymer plates. At the same time, hydroxyapatite particles in the matrices can affect the degree of osteogenic differentiation of cells. Also, the change in the percentage of polymers relative to each other affects both the proliferation and the degree of osteogenic differentiation of cells. However, despite this fact, the degree of proliferation and synthesis of the calcified extracellular matrix is lower than that of control cells.

Discussion

The development of biodegradable membranes for jawbone augmentation offers a promising alternative to traditional materials, such as titanium meshes. Our study focused on the use of PLA, PLGA, and HA in creating bioresorbable scaffolds and evaluated their effectiveness in promoting cell proliferation and osteogenic differentiation.^6,9 It was found that PLA and PLGA are biocompatible polymers with favorable properties for medical applications, including biodegradability and bioresorbability;^9,10,16 PLA has gained popularity in medicine due to its ability to naturally dissolve in the body, eliminating the need for secondary surgeries for material removal.

Titanium meshes have been widely used for oral and maxillofacial defect reconstruction due to their rigidity and ability to maintain the grafted space, preventing soft tissue collapse and enhancing bone regeneration. However, titanium meshes require intraoperative adaptation, which can prolong surgery time and increase the risk of complications, including mucosal irritation and the need for a second surgery to remove the mesh. Customization through CAD/CAM technology has improved the fit and stability of titanium meshes, reducing operation time and improving clinical outcomes.^17,18 Despite these advancements, the necessity of mesh removal remains a significant drawback.

In contrast, biodegradable materials, such as PLA and PLGA, offer the advantage of eliminating the need for a second surgery.¹⁴ These materials gradually resorb in the body, reducing the overall treatment timeline and associated risks. As mentioned, a variety of preclinical studies (hydrolytic degradation tests, enzyme resistance tests, bacterial collagenase resistance tests, mechanical tests, scanning electron microscopy, etc.) are required to confirm the ideal composition of biodegradable membranes that would meet the gold standard for bone tissue augmentation in the oral cavity. Our research demonstrates the results of one of the very first and important in vitro tests needed to assess the influence of materials on cells and their toxicity as well as the potential for osteogenic differentiation in the presence of various polymers. This stage is crucial as its outcomes can impact the further research strategy, determining the need for additional tests for full material approval. The results we have obtained confirm that biodegradable polymers, such as PLA and PLGA, are promising for maxillofacial surgery and require further steps.

In this study, we investigated the influence of different polymer compounds, including PLA, PLA/HA, PLGA 85/15, and PLGA 60/40, on the proliferation and osteogenic differentiation of gingival fibroblasts in vitro. Our findings indicate that cells cultured on composite matrices exhibited proliferation and differentiation toward an osteogenic lineage.^5,9 Notably, supplementation with hydroxyapatite particles enhanced the formation of calcified extracellular matrix, suggesting a potential role in promoting tissue calcification.⁶

Our study demonstrated that scaffolds made from PLGA (85:15 and 60:40 compositions) showed superior results in cell proliferation and matrix calcification compared with other materials tested. These findings align with previous research that highlights the biocompatibility and favorable degradation characteristics of PLGA. Furthermore, variations in the composition of polymer matrices appeared to affect both cell proliferation and osteogenic differentiation. For example, a change in the percentage of PLGA relative to PLA influenced the degree of calcified extracellular matrix formation.^4,8,9 These observations underscore the importance of optimizing membrane composition to maximize the stimulatory effect on osteogenesis.

Whereas our study observed the active proliferation of gingival fibroblasts at the edges of the matrices, indicating the biocompatibility of the materials, it is important to note that this alone is insufficient to conclude potential toxic reactions and the biodegradation time period. Further tests are essential to evaluate these aspects comprehensively.¹⁹

Two crucial tests that warrant consideration are the hydrolytic degradation and enzyme resistance tests. The hydrolytic degradation test involves analyzing the in vitro behavior of the membranes in PBS at 37°C. This test provides valuable insights into how the membranes degrade over time in a simulated physiological environment.¹² Similarly, the enzyme resistance test involves immersing the samples in a porcine trypsin solution and incubating them at 37°C. This test evaluates the resistance of the membranes to enzymatic degradation, which is pertinent considering the presence of various enzymes in biological tissues.²⁰

Biodegradable membranes present several clinical advantages over traditional nonresorbable materials.²¹ They reduce operation time as they do not require complex adaptation and are ready for passive fixation. This aspect is particularly beneficial for young dental professionals, broadening the demographic of patients who can receive such treatments. Additionally, the use of bioresorbable materials minimizes surgical risks by eliminating the need for a secondary procedure to remove the scaffold.²²

Our study’s results, showing active proliferation of gingival fibroblasts and enhanced calcification in the presence of PLA/HA and PLGA scaffolds, support their potential application in clinical settings. These materials demonstrated good biocompatibility and did not induce toxic reactions, which is crucial for their use in bone augmentation. Furthermore, the increased intensity of alizarin red staining in PLA/HA samples suggests that these scaffolds could promote better calcification of the extracellular matrix, essential for bone regeneration.

Despite the promising results, it is crucial to acknowledge this study’s limitations. The in vitro nature of the experiments may only partially reflect the complexities of bone regeneration in vivo. Further investigations are needed to evaluate these materials’ long-term performance and biocompatibility in animal models and clinical settings.

Conclusions

Supplementation of samples with hydroxyapatite particles enhances the formation of calcified extracellular matrix in gingival fibroblasts, indicating a potential role of hydroxyapatite in promoting tissue calcification. Assessment of osteogenic differentiation of gingival connective tissue cells on various composite matrices, including modified PLA, PLA/HA, PLGA 85/15, and PLGA 60/40, reveals that these cells exhibit proliferation and differentiation toward an osteogenic lineage when cultured on polymer plates. This suggests the suitability of these composite matrices for supporting osteogenesis.

Future investigations should optimize membrane composition to maximize the stimulatory effect on osteogenesis. Also, studying these materials’ in vivo bioresorption kinetics is crucial for understanding their long-term performance and biocompatibility.