Reinforcement of Oracle Bones Using a Novel Silicone Coupling Reagent for Preservation

Abstract

Oracle bones are artifacts of great significance and value in the study of Chinese history and culture. Because of soil and bacterial erosion, bones become fragile, and the inscriptions on the surface become blurred, resulting in the loss of historical information on the bones. In early times, scholars often used adhesives for bonding and reinforcement, whereas in modern times, organic and inorganic materials have been used as reinforcement for oracle bones. In this study, the surface of oracle bone was protected and reinforced by a new silicone coupling reagent that could self-polymerize in the format of colorless solution with good antimicrobial properties. The DESPMA was applied to the surface of oracle bone by dropwise addition and curing, effectively protecting it from bacteria and slowing down the yellowing process. The results showed that the reagent could significantly improve the antimicrobial properties of bone samples and reduce the yellowing and discoloration caused by bacterial attack. The reagent barely affected the appearance of the bone samples. These findings are promising and valuable for effective application in bone protection and utilization.

1. Introduction

A large number of bone products exist in historical artifacts, most of which are made from ivory, tortoiseshell, and animal bones. Artifacts made from tortoiseshell and animal bones are known as oracle bones. The bones used in oracle bones include pig bones, sheep bones, bovine bones, and a few human bones.

The writing recorded on oracle bones provides important evidence for studying history, but the process of making oracle bones is complicated. Ancient peoples cauterized and drilled these bones and applied writing to them, which documented the lives of the nobles of the Shang Dynasty [1,2,3]. This action makes oracle bones fragile and difficult to preserve [4,5].

Research has shown that the chemical composition of oracle bones includes both inorganic and organic parts [6]. Organic materials account for 30%, mainly including proteins and polysaccharides, and inorganic materials account for approximately 70%, mainly comprising calcium phosphate, carbonate, and fluoride. After thousands of years of soil burial, the non-apatite portion of the mineral composition becomes more unstable, and some inorganic materials are dissolved [7,8]. This results in varying degrees of cracking, fragmentation, and yellowing of oracle bones.

After many years of soil burial, some chemicals, bacteria, and fungi in the soil again change the morphology and chemical composition of oracle bones [9,10,11]. Figure 1 shows the oracle bone ZR044547 from a collection in Taiwan, China, which was embellished and pasted in 2010 [12]. The images show that, as a result of the corrosion of the soil (Figure 1A), the color distribution of the surface is uneven, and some of the oracle bones have begun to show a darker brown color.

However, this information holds great significance to the study of history, which provides rich information on rituals, diet, and diseases [13,14,15,16]. In order to protect the bones, scholars began to study the use of chemicals to protect and repair oracle bones [17].

The physical preservation of the oracle bone is mainly achieved through vacuum storage to protect the oracle bone [18]; Because some oracle bones are fragile and cannot be vacuum treated, scholars began to study the use of chemical adhesives. In previous studies, plaster or glue adhesives were usually used to repair the bones. Glue adhesives were made mainly from cellulose nitrate and cellulose acetate dissolved in acetone [19]. Figure 1B shows a large trace of glue.

In general, the protective agent needs to have good working properties and provide cohesion, strength, and durability [17,20]. In practice, scholars have tried to use a variety of materials for the protection of oracle bones, such as B-72 acrylic resin, polyvinyl acetate, polyvinylidenebutyl alcohol, polyethylene glycol, and acrylic reinforcement [21,22,23]. They also have adopted different techniques for the use of these reagents, such as dripping, spray painting, submerging, and decompression infiltration [24,25].

In the 1980s, a class of organic silicon anti-weathering materials, including the compounds of methyl trimethoxysilane, polysiloxane, and silicone were developed [26,27,28,29]. Polymer resin materials have been widely used in the restoration of cultural relics, such as type 509 epoxy resin which is often used in porcelain, and three, a resin used in metal cultural relics coating. At the same time, 2-hydroxyethyl methacrylate (HEMA), as the main monomer component in composite resin material matrix, is also widely used in the field of dental materials (self-etch adhesives and luting composites) [30,31]. Researchers are also exploring the antioxidant and antibacterial properties of composites made with HEMA as a monomer.

In this study, we proposed a novel organic silicon coupling reagent protectant for the prevention of bacterial attack on fragile oracle bones. Figure 2 schematizes the mechanism of action of the proposed reagent on the oracle bone. The first image shows a large gap inside the oracle bone, the second image shows the addition of reagents to fill the gap, and the third image shows the inhibitory effect on bacteria. Our goal was to reduce the negative consequences of discoloration and corrosion of the oracle bone after bacterial attack and to reduce preservation damage. We evaluated the antimicrobial effect of the agent with X-ray diffraction (XRD), scanning electron microscopy (SEM), Vickers hardness measurement (n = 2, 3 readings per specimen), antimicrobial properties, and penetration velocity measurements.

2. Materials and Methods

2.1. Reagents and Materials

2-Hydroxyethyl methacrylate (HEMA), saturated sodium chloride, and anhydrous sodium sulfate were purchased from National Pharmaceutical Group Co. (Beijing, China). Gelatin with the texture of colorless or light yellow flakes or lumps was produced by Zhonglian Chemical Reagent Co., Ltd. (Tianjin, China). DESPMA (2-(2-(Methacryloyloxy)ethyl)disulfide)ethyl(3-(diethoxymethylsilyl)propyl)carbamate) was synthesized according to a published study [32].

HEMA-CDI (10.07 g, 44.9 mmol) and 3-(diethoxymethylsilyl) propylamine (9.04 g, 47.24 mmol) were placed into a 100 mL round bottom flask, then 50 mL of dry dichloromethane (DCM) was added. After stirring the reaction mixture for 1 h, the DCM was removed and the residual oily substances were precipitated in petroleum ether twice. The obtained light yellow oily substance was dried under vacuum (yield: 92.9%) [33].

Real oracle bones are precious and their chemical composition would be unpredictably damaged by their use in this study. In general research, real oracle bones are not usually allowed to be used for experiments. According to the scientific experimental method, we used two materials in this experiment: cauterized bovine bone and simulated bone samples. The cauterized bovine bone simulated the production process of the oracle bone, and the simulated bone sample simulated the composition of the oracle bone [34].

2.2. Bovine Bone Samples

It has been found that the oracle bones burn at different temperatures and, if soaked with acid after excavation, would be weathered [35]. High temperatures, drilling, and chiseling operations will change the physical structure and chemical composition of oracle bone [36]. To better verify the effect of the reagent, we used cauterized bovine bone in the experiment. To prepare the bovine bone, the following steps were used:

• The bones were boiled for 1 h in a solution of sodium bicarbonate to remove oil and grease;
• The cooled bones were dried in the air for 2 d and then sawed into rectangles of similar size by using a Low Speed Diamond Saw (SYJ-150, Shanghai Optical Instrument Factory, Shanghai, China);
• The cut bones were placed in a muffin pan, burned at 550 °C for 4 h and then cooled for 8 h;
• The resulting bones were soaked in 4.5% hydrochloric acid for 5 min, rinsed with water three to four times, and then dried in the air for 2 d.

Figure 3 shows the bovine bone samples before (Figure 3A) and after being treated by boiling in sodium bicarbonate solution and drying at room temperature (Figure 3B), and by firing at high temperature (550 °C) (Figure 3C). The treated bone exhibited a whitening effect, a decrease in bone quality and surface curvature, and/or a tendency to release white powder on contact. These negative effects seriously affected the integrity of the bone surface.

Figure 4 shows the XRD results of the real oracle bone and bovine bone samples. When the real oracle bone was tested, the diffraction peaks of gelatin appeared at 25.91°, 31.84°, 39.49° and 48.59° (Figure 4A), which was consistent with the hydroxyapatite diffraction data in the database. The bovine bone sample (Figure 4B) showed diffraction peaks that almost identical to those of the real oracle bone.

2.3. Simulated Bone Samples

The composition of oracle bone will change after thousands of years of burial. To better verify the effect of the reagent, we used a simulated bone sample in the experiment. The simulated bone samples were prepared based on the percentage content of different metacarpal bone substances, as shown in

Table 1. Percentage chemical composition of oracle bone substances.

Water	Organic Matter	Carbonate	Soils	Hydroxyapatite
2.40	8.24	3.13	13.56	72.68

[5]. Then, 0.23 g of water, 0.79 g of gelatin, 0.3 g of calcium carbonate, 1.3 g of undried and unsterilized earth, and 6.97 g of hydroxyapatite were mixed. The mixture was stirred in an agate mortar and divided into five equal portions of ~2 g per sample. Each portion was pressed into a powder tablet at 1 MPa by using SZT-15T manual powder tablet press (Tianjin Zhongtuo Science and Technology Development Co, Tianjin, China).

Silica resin contains a hydrophobic alkyl group and a silicon oxide group for the formation of the Si-O-Si network. It has the flexibility of organic materials, water resistance, and the aging resistance of inorganic materials. Research shows that the silicone resin has good permeability and combined strength to strengthen the internal structure of the overall mechanical strength and aging resistance of the relics.

Scanning electron microscopy (SEM, FEI SIRION-100, 5.0 kV of accelerating voltage, Hillsboro, OR, USA), Fourier transform infrared spectroscopy (FTIR, Thermo Scientific™ Nicolet™ iN10, 4000–450 cm⁻¹ range, Thermo Fisher Scientific, Madison, WI, USA), and X-ray diffractometry (XRD, AXS D8 ADVANCE, Cu Ka radiation, scan range 2θ = 10–80°, Bruker, Billerica, MA, USA) were used to analyze the structure and composition of the samples.

2.4. Plate-Coated Colony Counting Assay

The bovine bone samples treated by DESPMA were used as carriers for the autoclaved materials. The antimicrobial properties of the samples were verified using colony counting on agar plates. The experimental samples and equipment were sterilized before the experiment. All the samples were sterilized by ultraviolet disinfection, and the sterilized samples were put into disposable plates to characterize the antibacterial properties of the surface.

• 500 g of soil was collected, soaked in deionized water, covered with the soil surface for 2–5 cm, stirred and settled overnight, and an appropriate amount of supernatant was taken as the source of the flora for this characterization experiment.
• Sample A, treated by DESPMA, had a solution of soil dropped onto on the surface. As the liquid needs to wrap around the sample surface as much as possible, the liquid was spread as far as possible.
• The sample B, without the DESPMA, had a solution of soil dropped onto the surface. As the liquid needs to wrap around the sample surface as much as possible, the liquid was spread as far as possible.
• The incubation temperature was 37 °C, the humidity was 60%–98%, and the incubation time was 24/48/72 h.
• After incubation, 10 (L medium) was taken from the surface of samples A and B. The solutions were coated in three gradients and counted separately, namely, 1×, 10× and 100×.

3. Results

To improve the precision of the permeability at the molecular level, a new component, a reducible silica monomer called DESPMA, was used. The molecular structure of the reagent along with its ¹H nuclear magnetic resonance spectroscopy (¹H NMR) data are shown in Figure 5. We perform integration on specific peaks during the process. The integrated areas of the two are in a ratio of 4:6, which demonstrates that the two raw materials are coupled in a 1:1 ratio. The diethoxymethylsilyl property of DESPMA by in situ crosslinking caused a lower crosslink density and a greater permeability [32].

The methacrylate group in this monomer was used for free radical polymerization, the disulfide bond has a certain reduction response, and the two alkoxygen groups connected to the silicon atoms were used for crosslinking curing.

As shown in Scheme 1, DESPMA first penetrated into the oracle bone, and then triggered the crosslinking reaction, playing a role in reinforcement and protection.

From the infrared spectrum of DEPMA, it can be observed that, the stretching vibration absorption peak of N-H is at 3340 cm⁻¹, the stretching vibration absorption peaks of C-H are at 2973 cm⁻¹ and 2881 cm⁻¹, the stretching vibration absorption peak of the ester group C = O is at 1719 cm⁻¹, the stretching vibration absorption peak of the C = C double bond is at 1527 cm⁻¹, the bending vibration absorption peak of-CH₂-is at 1444 cm⁻¹, the bending vibration absorption peak of-CH₃ is at 1390 cm⁻¹, the stretching vibration absorption peak of C-O is at 1244 cm⁻¹, the in-plane bending vibration absorption peak of C-H is at 1164 cm⁻¹, the out-plane bending vibration absorption peak of C-H is at 942 cm⁻¹, and the antisymmetric stretching vibration absorption peaks of Si-O are at 1072 cm⁻¹ and 763 cm⁻¹ (Figure 6).

The penetration ability of the reagent is a key factor for the effect of the reagent, and the penetration ability determines the area of the reinforced oracle bone.

In order to better improve the permeability, the density of Si-O-Si bonds was reduced, and the silicon atoms act in only two directions that can be connected to the crosslinked network. The remaining two directions are not connected to the crosslinked network. The introduction of silicon oxide groups (Si-O-Si) can change the size of the pores in the network. By adjusting the number and distribution of silicon oxide groups, the pore size precisely controls the permeability of the material. Larger pores favor the diffusion and penetration of macromolecules or hydrophobic molecules.

3.1. Micro-Structure Evolution of the Simulated Bone Samples

Scanning electron microscopy (SEM) was utilized to investigate the evolution of the microstructure of the samples. The magnification was adjusted to 200 μm and 5.00 μm to meticulously analyze the surface features. As shown in Figure 7A,C, the untreated samples present a rough and porous surface texture. In contrast, the samples treated by DESPMA (Figure 7B,D) exhibit a smoother surface. The reagents fill in the pore to some extent, thereby reinforcing and protecting the sample.

3.2. Surface Hardness Change

The reinforcement performance of the reagent was measured using Vickers hardness. For two samples measuring 2 cm × 2 cm, one was untreated, and the other one treated by DESPMA. The specimens were placed in a microhardness indenter (Micromet 5100, Buehler, Singapore). Three indents were made in each specimen, near the center of the specimen and at least 0.5 mm away from each other. As shown in Figure 8, the hardness of sample b increased by 6.8% to 26.8%. To increase the reliability of the experiment, by averaging the three values, the hardness of sample B was increased by 14.3%.

3.3. Consolidation of Reagents

The consolidation of the adhesive is a key factor in oracle bone reinforcement. Because of their low boiling point, all solvents used in this article can evaporate in an open environment at room temperature. In addition, the consolidation speed can affect the applicable range and condition of the material to a large extent.

Two reagents, B72 (acrylic resin adhesive) and PS (liquid sodium silicate) were experimentally tested and compared with the present reagent. As shown in Figure 9, to make the experiment comparable, all materials were applied to the bone sample surface only once, and the mass changes were recorded immediately after the application. Reagent addition was followed by rapid volatilization and then a slow consolidation reaction. The consolidation rate for DESPMA (Figure 9C) is slightly lower than that of B72 (Figure 9B) but higher than that of PS (Figure 9A).

To investigate the possible mechanism of the change above, the composition of the bone samples was monitored during the consolidating process by FTIR analysis. The infrared spectrogram of the bovine bone sample reveals distinct absorption peaks corresponding to specific vibrational modes. At 1413 cm⁻¹, an asymmetric stretching vibration absorption peak is attributed to the carbonate ion (CO₃²⁻). Similarly, at 1028 cm⁻¹, an asymmetric stretching vibration absorption peak is indicative of the P-O bond. The symmetric stretching vibration of the P-O bond is observed at 962 cm⁻¹, while the symmetric stretching vibration of the carbonate ion (CO₃²⁻) is discernible at 873 cm⁻¹. Furthermore, the stretching vibration of the Ca-O bond is characterized by an absorption peak at 631 cm⁻¹.

The N-H group (at 3340 cm⁻¹) within DESPMA may engage in hydrogen bonding with the phosphate (P-O) or carbonate (CO₃²⁻) groups present in the bovine bone. This interaction can enhance the cohesion and increase the hardness of the sample, without generating any new chemical absorption peaks in the infrared (IR) spectrum. The Si-O groups in DESPMA (1072 cm⁻¹ and 763 cm⁻¹) may form a silica bridging bond with the hydroxyl group or water molecules in the bovine bone, which helps to improve the crosslinking density and stability of the material (Figure 10).

3.4. Volatility of Reagents

The volatilization speed of the solvent in the reinforcement material has a great impact on the final protection effect. A fast volatilization speed may reduce the depth of the reinforcement agent, and even cause a reverse migration phenomenon of the reinforcement agent, thus resulting in secondary damage. Therefore, we tested the volatility of the solution.

We also used a circular simulated bone sample of the same diameter in this experiment. After complete coverage of the entire surface with the reagent, the size of the area covered by the liquid on the surface of the bone sample was determined and the time it took for the reagent to completely vaporize was recorded. The bone sample had a diameter of R and an area of πR², and the reagent covered an area with a diameter of r and an area of πr². The percentage of the area covered, E, was calculated as follows in Equation (1):

$$E=r^2/R^2,$$

(1)

As shown in Figure 11, DESPMA evaporated faster when the reagents were applied to the bone surface, avoiding chemical damage to the surface of the oracle bone to a greater extent and minimizing influence on surface color and appearance. The curve will eventually become zero, since the solvent of the reagent is volatile.

3.5. Reagent Protection

Heat resistance is one of the important performance indexes of silicone coating. We also performed another test by dropping the same solution on a slide coated with hydroxyapatite gelatin and placing it in an oven at 40 °C. The breakage and peeling of the slides were recorded. In this experiment, the hydroxyapatite gelatin solution was added dropwise to the slides and it solidified to form a white solid-mounted flake covering the entire slide; then, 2 mL of water, PS, B72, and DESPMA solutions were added dropwise to each of the four slides. After dying at room temperature, the slides were heated in an oven at 40 °C. The time until the cracking of hydroxyapatite gelatin was recorded. As shown in Figure 12, the sample treated with an aqueous solution cracked after only 15 min of heating in the drying oven. The protection time was extended to 34 min for the sample treated with B72 solution, 45 min for the sample treated with PS solution, and 48 min for the sample treated with DESPMA solution. This result can be attributed to the high bond energy of the Si-O bond (~460 kJ/mol) [37] that endows the reagent with excellent heat resistance and chemical stability.

3.6. Effects of Bacteria on Agar Plates

Figure 13 shows the petri dish coated with the solution of soil. Figure 13B shows a yellow bacterial colony, identified as Staphylococcus aureus. The next day, we observed multiple colonies with white and yellow dots on the petri dishes, indicating a relatively high concentration of microorganisms in the solution.

Samples were taken every 24 h and coated at a multiple dilution of 1, 10 and 100, where (a) (b) (c) is the sample treated by DESPMA, and (d) (e) (f) is the untreated sample. Figure 14A shows that the number of colonies on ×100-fold diluted (c) plates was 461 and on (f) plates was 1121, decreasing by 58.8%. Figure 14 shows that DESPMA inhibited bacterial growth to some extent.

3.7. Effects of Bacteria on the Bone Samples

The simulated bone samples and bovine bone samples treated by DESPMA were used as carriers for the autoclaved materials. A series of tests was conducted to determine the effectiveness of the different solutions in preventing yellowing and damage of the bovine bone and simulated bone samples. To do this, we pipetted 2 mL each of B72, PS, and DESPMA solutions onto the samples and allowed them to cure. The chromatographic solutions were then applied uniformly to the surface and any changes in appearance were recorded, The numbers of yellow patches and microflora on the samples were examined by microscopy and counted. This rigorous procedure ensured the accuracy and reliability of our results.

As the results in

Table 2. Bacterial colonies counted on simulated bone samples.

Solution	Day 1	Day 7	Day 14	Day 21	Day 28
DESPMA	0	0	0	2	4
PS	0	0	2	5	6
B72	0	0	2	4	5
Water	0	0	3	5	8

and

Table 3. Bacterial colonies counted on for bovine bone samples.

Solution	Day 1	Day 7	Day 14	Day 21	Day 28
DESPMA	0	0	2	3	5
PS	0	3	5	7	8
B72	0	2	4	6	8
Water	0	3	3	7	9

show, the bone samples treated by DESPMA developed fewer bacterial colonies. Using day 28 as a reference, there was the lowest number of colony counted on the surface of the bone samples treated by DESPMA, and a relatively good performance in both the simulated bone samples and the bovine bone samples. The degree of yellowing on the bovine bone samples increased only slightly compared with the simulated bone samples, highlighting the effectiveness of the DESPMA solution in preventing yellowing.

4. Conclusions

The physical protection of oracle bones is crucial. In this study, we proposed the use of a chemical method to protect the surface of oracle bones from erosion. Specifically, we successfully developed a new silicone resin reagent (DESPMA). The resulting product was then dissolved in dichloromethane to form a reinforcer solution with superior antimicrobial and penetration properties. We applied droplets of the solution to the surface of the oracle bones to test its effectiveness as a protective material. After the curing treatment, the bone samples showed improved protection and antimicrobial properties, and the change in appearance was within acceptable limits. This study proposed a new means to protect oracle bones, although the reagents used should be further refined so that they can provide better protection for the information on the bone surfaces by strengthening the internal physical structure of oracle bones.