Research on functional boards and plastic lms based on animal skin and hydroxyapatite

Nano-Hydroxyapatite precursors with calcium and phosphorus salts (Ca/P = 1.67) were introduced into a three-dimensional collagen network matrix (3DCM) based on goat skin pretreated with glutaraldehyde for in situ growth, and a functional HAG-3DCM board was obtained. After plasticity compression, a transparent protein plastic lm was formed. Response surface methodology based on plasticity pressure conditions was used, and the strength, hardness and water resistance of the HAG-3DCM plastic lm was signicantly improved. This study demonstrates a new approach for the preparation of animal skin materials with new application value.


Introduction
The use of animal skins has a long history, and even 8000 years ago, animal glue from animal skin extracted by cavepeople in the eastern region was used as a home decoration adhesive. Gelatin from animal skin has played an important role in photography, medical accessories, drug packaging, the food industry and so on. [1][2][3] Tanning leather represents the best use of animal skin, and leather products were found in Egyptian pyramids 3,000 years ago. 4 Animal skin is composed of three parts, namely, the epidermis, dermis and subcutaneous tissue. The main structural component of the dermis is a protein collagen. After the removal of water, inter brillar substance and fat, collagen accounts for over 95.0% of the remaining material, most of which is type I collagen, and a small amount is type III collagen. 5 As a natural renewable resource, protein has become a new research focus as an alternative to petroleum as a aky material in the eld of decorative boards and packaging lms. Biobased plastics made of protein or "protein plastics" have the characteristics of low cost, nonbiological toxicity, good degradability. 6 Protein plastics mainly come from soybean protein, cottonseed protein, corn protein and wheat protein. [6][7][8] Because protein generally has inherent defects including poor water resistance, low mechanical properties, poor processability, etc., it is usually used as a ller or additive to blend with ordinary petroleum-based plastic materials, which can reduce the cost of plastic materials and promote plastic decomposition to reduce environmental pollution. 9,10 Hydroxyapatite (HA) is slightly soluble in water and weakly alkaline (pH=7.0~9.0), and it has excellent biological activity, biocompatibility and nontoxicity; it is widely used in bone repair, bone transplantation and drug transportation. 11 Natural bone is a composite material composed of type I collagen and HA. HA accounts for 70.0% of the backbone mass and 90.0% of the remaining 30.0% organic components are type I collagen. Arti cial bone preparation involves adding calcium and phosphate salt solutions to the collagen solution in sequence, and the obtained HA particles are evenly distributed in the collagen matrix or embedded on the surface of the collagen molecule. This type of synthesis method is also called the in situ synthesis law. 12 A large number of research reports have found that HA/collagen composites generally have shortcomings such as low mechanical strength, easy swelling, and poor inducing activity, 12 which limits the application channels. In this paper, HA was introduced into natural three-dimensional skin collagen pretreated with glutaraldehyde to induce in situ aggregation, crystal growth and collagen tissue mineralization, 13 which hardened the collagen matrix and improved the thermal stability; 14,15 the heat insulation and ame retardant capacities of the composites were measured.
Further, the preparation of the protein-based plasticity compression lm was studied, 16,17  Softened goat skin collagen, was obtained from a number of New Zealand salted sheep skins with an area of 0.7~1.0 m 2 , and it was prepared and puri ed by removing the noncollagen components, including the hair, epidermis, grease, and inter brillar substances. 18 Finally, the pH of the skin collagen was adjusted to 4.5~5.0 with 2 mol/L ammonium sulfate, and the puri ed three-dimensional collagen ber material matrix (3DCM) with a thickness of 0.65±0.05 mm was obtained by 10 kg of centrifugal dehydration.

Preparation of micronucleus precursor HA in vitro
Then, 1000 mL of deionized water and 200 g of calcium nitrate and diammonium hydrogen phosphate mixture (according to Ca/P=1.67) were added to the three-necked ask. The pH value of the solution was adjusted with ammonia to 11.0, and the solution was stirred at 80°C for 12.0 hours and allowed to stand for 12.0 hours. The solution was centrifuged at 10.0 kg, and the precipitate was dried to a constant weight at 105°C for sample analysis.

Penetration and growth of precursor HA in 3DCM
3DCM (by dry weight) was weighed and put into a Ф600 drum (Derun Light Industry Machinery Factory, Wuxi, China), and then, 150% (30°C) water and 2.0% glutaraldehyde (by effective content) were added, rotated for 120 min, stopped after 120 min, and rotated again 5 times. Subsequently, the pH was adjusted to 6.0 ~ 6.6 with 10% NaHCO 3 solution within 60 min, and then, the liquid was drained. Water (150%) was added, and then, the solution was rotated for 10 min at 50°C to obtain a 0# sample (3DCM). Based on the 3DCM sample, 5% micronucleus precursor HA and (1~4)×(4.0% Ca(NO 3 ) 2 +2.8%((NH 4 ) 2 PO 4 ) were added according to the Ca/P of 1.67 (note 1#~4#). After rotating at 5 r/min for 4.0 hours, the solution was allowed to stand for 48.0 hours, and then, it was dried naturally and stretched to obtain, 1#HAG-3DCM~4#HAG-3DCM samples. The optimized HAG-3DCM sample was calcined in a mu e furnace at 800°C for 2.0 h to remove organic components, and then the calcination residual was washed with water to remove soluble inorganic components. After drying, an X-ray diffractometer (X'PertProMPDY129, Nalytical, Netherlands) was used for the remaining inorganic phases. The construction of outsourced biograde HA and HA in the optimized HAG-3DCM was analyzed and compared. The selected scanning angle step was 0.02°, the scanning range was 10.0 ~ 70.0°, and the scanning speed was 2°/min.

Morphology of precursor HA and HA in HAG-3DCM
To observe the morphology of precursor HA and HA in the optimized HAG-3DCM, the precursor HA and the optimized HAG-3DCM samples were calcined in a mu e furnace at 800°C for 2.0 h, and then, two calcined residuals were washed with water to remove the organic and soluble inorganic components. After drying, TEM analysis was performed on two calcined residuals that were xed on the sample platform with conductive adhesive, and the surface morphology was observed by a Tecnai G2F20 microscope (S-Twin, FEI Corporation, USA).

Element distribution characteristics in EDS
To understand the penetration and accumulation growth of HA precursors in 3DCM, EDS scanning was performed on the 3DCM cross-section at selected points based on the phosphorus-rich region and scanning was carried out at a diameter of 0.005 mm by energy dispersive X-ray spectroscopy (EDS, JSM-7500F, Japan Electronics Co., Japan).

Morphology of HAG-3DCM
The morphology of collagen ber bundles in the optimized HAG-3DCM was observed by scanning electron microscopy (JSM-7500F, JEOL Corporation, Japan). The test surface was sprayed with gold, and the acceleration voltage was 3.00 kV.
2.6 Functional characterization of HAG-3DCM plate materials 2.6.1 HAG-3DCM thermal insulation test The thermal conductivity of the optimized HAG-3DCM was characterized by the steady-state method to measure the thermal conductivity of the sample. Using the coe cient of the thermal conductivity tester (DRP-, Xiangtan Xiangjiang Instrument Co., Ltd.). First, the sample was placed between the upper and lower copper plates and heated to the set temperature. When the temperature of the upper and lower copper plates was stable, the temperatures T 1 and T 2 were recorded. Next, the sample was removed, the copper plate was heated to T 3 (higher than T 2 ), heating was stopped, and then, the change in temperature of the lower copper plate over time was recorded again.
where m is the mass of the lower copper plate, g; c is the speci c heat capacity of the copper plate, kJ/(K·kg); h p and R p are the thickness and radius of the lower copper plate, respectively, mm; h is the sample thickness, mm; and T 1 and T 2 are the temperatures of the upper and lower copper plate, respectively, °C. The basic diagram is shown in Figure 1.

Flame retardant test of HAG-3DCM
The optimized HAG-3DCM was tested according to GB/T2406-1993 (Plastics Combustion Performance Test-Oxygen Index Method). An oxygen index tester (JF-3, Nanjing Jionglai Company) was used to test the limiting oxygen index of the samples. The combustion time was 15.0 s and the combustion length was 5.0 cm.

Test of the physical and mechanical characteristics of HAG-3DCM
Tested samples of the optimized HAG-3DCM were cut and kept at constant temperature (25.0°C) and humidity (65.0% RH) for 24 h. According to the standard QB/T2710-2005 the tensile strength and elongation at break of the samples were measured by using a universal tension machine (AL7000SN, Taiwan High-speed Railway Technology Company). At 25°C, the rising rate of the instrument was 50 mm/min. Each group of samples was measured in 5 parallel tests, and the average value was taken. where S is the swelling degree (%), M is the initial mass (g), and M S is the swelling mass (g).

Light transmittance test of the 3DCM-PP and HAG-3DCM-PP
The 3DCM-PP and HAG-3DCM samples were cut to a size of 2.0×5.0 cm. The transmittance of the samples in three different locations was measured by a light transmittance tester (LS116, Shenzhen Lianhuicheng Technology Co., Ltd.) in the length direction, and the average values were obtained.

Flexural resistance of the 3DCM-PP and HAG-3DCM-PP
According to the standard of leather torsion fastness (QB/T 2714-2018), constant temperature (25.0°C) and constant humidity (65.0% RH) conditions were applied for 24.0 h, and then, the 3DCM-PP or HAG-3DCM-PP lms were placed in a torsion fastness tester (XK-3014, Jiangsu Xiangke Instrument Company, China). The lm size was 70×45 mm and the winding angle was 22.5°. The maximum torsion fastness was observed by 5 cycles until cracks were observed under 5× magni cation.

Optimized HAG-3DCM
The analysis results of the complex formed by HA growth in the 3DCM sample are shown in Table 1. Ca and P determined by ICP after digestion of HAG-3DCM and w(HA)/% were calculated according to a Ca/P of 1.67. The results show that 0#3DCM contains negligible amounts of HA and 1#~4#HAG-3DCM are close to the Ca 10 (PO 4 ) 6 (OH) 2 structure in 3DCM. The Ca/P ratios in 1#~4#HAG-3DCM are all higher than 1.67 because there was a small amount of Ca in 3DCM, which was derived from the preparation process of 3DCM. Table 1 shows that the addition of Ca(NO 3 ) 2 and (NH 4 ) 2 PO 4 in 4#HAG-3DCM compared with 3#HAG-3DCM increases by 25%, although the effective binding w(HA)/% increases by 0.08%. The Ts values of 1#~4# HAG-3DCM are mainly from glutaraldehyde tanning; thus, the difference among them is small, but the 3# is much closer to the 4# Ts. Therefore, 3#HAG-3DCM can be considered to have reached the stabilization effects, turning point and represents the optimized process for the preparation of 3DCM in this experiment. Hereafter, this variant is called HAG-3DCM in the experimental samples.

Element distribution characteristics in HAG-3DCM
Since almost no P is observed in 3DCM, it represents the aggregation point of HA in the phosphorus-rich region. Four HA growth points were identi ed by scanning from the 3DCM surface in an approximate range of 0.7 mm. The elemental analysis is shown in Figure 2. According to the analysis of the surface scanning results, the Ca/P in HA-3DCM was 1.71~1.80, which was larger than that of standard HA. When the pH is higher than the pI of 3DCM, the penetration and combination of Ca 2+ in 3DCM is easier while that of PO4 3is impeded; thus, Ca/P can not meeting the theoretical value of 1.67.

HA morphology in 3DCM
The two calcination residuals from the precursor HA and HA in the HAG-3DCM sample were observed by TEM, as shown in Figure 3. Compared with the HA from the precursor, the HA from HAG-3DCM was similar to the precursor HA in a columnar shape, although the diameter of the precursor HA was less than 100 nm, and HA particles in the HA-3DCM sample were more bonded and slightly larger in volume.
Therefore, crystal growth occurs in the 3DCM sample, as shown in Figure 3.

HA structure in HAG-3DCM
The XRD spectra of standard HA 19 and inorganic phase samples in the HAG-3DCM sample were compared, as shown in Figure 4. The main diffraction peaks of the inorganic phase in the body correspond to the characteristic diffraction peaks of the standard HA in Figure 4, in which the characteristic peaks (002), (210), (211), (300) and (213) appear in turn. It was further con rmed that the inorganic phase in the HA-3DCM sample was HA.

Formation process of HAG-3DCM
According to the preparation process of HAG-3DCM and the morphology and structure of HA, the crystal growth process of HA in 3DCM is shown in Figure 5.

HAG-3DCM ber bundle braiding
By comparing the SEM images of the cross-section and longitudinal section of raw material 3DCM and compound HAG-3DCM. As shown in Figure 6, the formation of HA in the 3DCM caused the HAG-3DCM ber bundles to separate from the aggregates, which is consistent with the mechanism of the separation and xation of skin collagen tissues by tanning agents in the tanning chemistry. Thus, HAG-3DCM can be considered a new type of chrome-free tanned leather.

Heat insulation of HAG-3DCM
The thermal conductivity of 3DCM and the HAG-3DCM lm was measured by the steady state method at 60°C, as shown in Table 2. The thermal conductivity of HAG-3DCM is decreased from 0.25 W/K·m to 0.12 W/K·m compared with that of 3DCM. The decrease in thermal conductivity is caused by cavitation (see Figure 5) of the collagen matrix after 3DCM is treated with HA, and the increase in porosity leads to a decrease in thermal conductivity. 20 In the combustion process, a carbonization layer is formed on the surface of the 3DCM and HAG-3DCM samples. The many internal pores in HAG-3DCM can store the generated ammonia nitrogen and block the oxygen and external heat sources to prevent further combustion. 21 Table 2 shows that HAG-3DCM has a relatively high limiting oxygen index of 26.5%. Table 3 shows the physical and mechanical values of the HAG-3DCM. Due to the lack of a standard comparison, the sample was used as a type of leather for reference in this experiment. Compared with the national standard of chrome-free upper leather (≤1.0 mm), it can be seen that except for the bursting force, the other indexes such as the tensile strength, tearing strength, and load elongation all meet the requirements. Tensile strength is related not only to the strength of a single ber, but also to the number of bers per unit cross section. The greater the strength of a single ber and the greater the number of bers per unit cross section, the greater the tensile strength. For a protein-based structure, as the density increases, the binding between molecules and molecular chains is strengthened until reaching a limit.

Physical and mechanical characteristics of HAG-3DCM
A response surface analysis was performed on the tensile strength test results of the sample with a thickness of 1.0±0.12 mm, as shown in Figure 7. When the molding time was 11.0 min, the tensile strength of the protein plastics increased with increasing pressure. When the molding time was 11.0 min, the increase in temperature had little effect on the tensile strength of the sample.
According to the results shown in Figure 7, Table 4 integrates the analysis of pressure, temperature and time factors and obtains the order of in uence as follows: pressure > temperature ≈ time. For 3DCM, in addition to the pressure factor, the in uence of the time factor is greater than that of the temperature because when the temperature is higher than 100°C, the 3DCM is subjected to thermal denaturation and gelation, which easily results in a rapid increase in the density of the 3DCM-PP and a change in the tensile strength because the rheological velocity becomes a major factor.
For the HAG-3DCM-PP, the temperature factor is greater than the time factor. Although the entry and growth of HA greatly improves the gelling resistance and inhibits the rheological rate after gelation of HAG-3DCM, the gelling degree of the HAG-3DCM-PP is greatly affected when the temperature is over 100°C.
The optimal test conditions for the 3DCM-PP and HAG-3DCM-PP and the tensile strength of the samples are analyzed according to the response surface, as shown in Table 5. The tensile strength of the HAG-3DCM-PP is better than that of the 3DCM-PP and exceeds 41.0 MPa. This indicates that HA aggregates and grows in 3DCM, which can mineralize collagen bers well, stabilize the structure of 3DCM, and signi cantly increase the tensile strength of the plastic lm. For the two kinds of lms, the optimum conditions that have common signi cance are a pressure of 10.5 MPa, temperature 135°C and time of 11.0 min.  Table 6. After HA enters 3DCM to form HAG-3DCM, a special stabilization effect or mineralization effect occurs, under which tensile strength and hardness are increased and the water swelling resistance is greatly improved. Although the transparency of 3DCM is higher than that of HAG-3DCM (as shown in Figure 8), the pores between the bers in HAG-3DCM can be removed by plasticity pressure to satisfactorily improve its transparency. According to the comparison of the exural resistance in Table 6, without HA support, glycerol could not improve the tortuous resistance of 3DCM. The data shown in Table 6 show that 3DCM has potential application value.

Conclusions
When nano-HA precursors with calcium and phosphorus salts (Ca/P=1.67) were in ltrated into the threedimensional collagen network matrix (3DCM) based on goat skin pretreated with glutaraldehyde for insitu growth, an ecological animal skin function board called HAG-3DCM was obtained. The collagen tissue was xed or mineralized in the form of dispersion or cavitation. After plasticity compression a transparent protein plastic lm (HAG-3DCM-PP) was formed. For the optimization of plasticity pressure conditions, the strength, hardness and water resistance of HAG-3DCM were signi cantly improved compared with the 3DCM (without HA). This study demonstrates a new approach to the preparation of ecological materials from animal skins with new application value.

Figure 1
Principle of the heat conduction test  In vivo growth process of HA Figure 6 SEM image of the collagen matrix before and after HA treatment