Analytically pure diammonium hydrogen phosphate ((NH4)2HPO4), calcium nitrate (Ca(NO3)), glycerol, etc., and biological grade hydroxyapatite (HA), were obtained from Chengdu Kelon Chemical Reagent Factory. Industrial grade polyurethane emulsion, was obtained from Xuzhou Hongfeng Polymer Material Company.
Softened goat skin collagen, was obtained from a number of New Zealand salted sheep skins with an area of 0.7~1.0 m2, and it was prepared and purified by removing the noncollagen components, including the hair, epidermis, grease, and interfibrillar substances.18 Finally, the pH of the skin collagen was adjusted to 4.5~5.0 with 2 mol/L ammonium sulfate, and the purified three-dimensional collagen fiber material matrix (3DCM) with a thickness of 0.65±0.05 mm was obtained by 10 kg of centrifugal dehydration.
2.2 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 flask. 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.
2.3 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% NaHCO3 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(NO3)2+2.8%((NH4)2PO4) 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.
2.4 Optimization of the HAG-3DCM sample preparation
2.4.1 Determination of the HA content and Ca/P in the samples
Certain amounts of the 0#3DCM and 1#HAG-3DCM~4# HAG-3DCM samples were dried at 105°C to a constant weight. The solution was composed of V(HNO3):V(H2O2) at 5.0:2.0. It was digested at 100°C for 2.0 hours and diluted by a certain multiple. Then the contents of Ca and P in the digested samples were measured by ICP (VG PQExCell, TJA Company, USA). The Ca/P ratio and HA content in the samples were calculated.
2.4.2 Moisture and heat stability test of the samples
Shrinkage of collagen is a dynamic process. The shrinkage temperature (Ts) reflects the difference in moisture and heat resistance of treated collagen. The Ts values of the 0#3DCM and 1#HAG-3DCM~4#HAG-3DCM samples were measured with an SMW-YD1 shrink temperature meter.
According to experiments 2.4.1 and 2.4.2, the optimized HAG-3DCM and its preparation process can be determined.
2.5 Characteristics of the optimized HAG-3DCM
2.5.1 HA structure test of HAG-3DCM
The optimized HAG-3DCM sample was calcined in a muffle 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.
2.5.2 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 muffle 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 fixed on the sample platform with conductive adhesive, and the surface morphology was observed by a Tecnai G2F20 microscope (S-Twin, FEI Corporation, USA).
2.5.3 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).
2.5.4 Morphology of HAG-3DCM
The morphology of collagen fiber 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 coefficient of the thermal conductivity tester (DRP-Ⅱ, 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 T1 and T2 were recorded. Next, the sample was removed, the copper plate was heated to T3 (higher than T2), 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 specific heat capacity of the copper plate, kJ/(K·kg); hp and Rp are the thickness and radius of the lower copper plate, respectively, mm; h is the sample thickness, mm; and T1 and T2 are the temperatures of the upper and lower copper plate, respectively, °C. The basic diagram is shown in Figure 1.
2.6.2 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.
2.6.3 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.
2.7 Functional characterization of HAG-3DCM plastic films
2.7.1 Preparation of plasticity pressed films
3DCM and optimized HAG-3DCM samples were cut into small pieces of 10×20 cm and uniformly coated with 100 g/m2 glycerin on both sides. The samples were dried completely in a vacuum at 35.0°C, and then kept at constant temperature (25.0°C) and constant humidity (65.0% RH) for 24.0 h. The samples were placed in the die of a hot press, which was designed according to the response surface test. The molding temperature was set at 100, 130 and 160°C, the pressure was set at 8.0, 10.0 and 12.0 MPa, and the molding time was set at 5, 10 and 15 min. The plasticity pressed samples were referred to as 3DCM protein plastic (3DCM-PP) and HAG-3DCM protein plastic (HAG-3DCM-PP).
2.7.2 Hardness test of the 3DCM-PP and HAG-3DCM-PP
According to the GB 2411-1980 method, the 3DCM-PP and HAG-3DCM-PP samples were cut with a sampler. A shore hardness tester (LX-A-1, Wenzhou Weidu Electronics Co., Ltd.) was used to apply 1.0 kg of load and pressure for 30.0 s, and the Shore hardness value of each sample was recorded.
2.7.3 Strength test of the 3DCM-PP and HAG-3DCM-PP
According to the GB/T1040-92 plastic tensile property test method, the 3DCM-PP and HAG-3DCM-PP samples were cut with a sampler. The tensile strength and elongation at break of the samples were measured by universal tensile machine (AL-7000SN, High-speed Rail Technology Co., Ltd.), and the tensile rising rate was 50.0 mm/min. Each group was measured in parallel 3 times, and the average values of the tensile strength and elongation at break of each sample were recorded.
2.7.4 Swelling property test of the 3DCM-PP and HAG-3DCM-PP
3DCM-PP and HAG-3DCM samples of the same amount were placed into Petri dishes and immersed in 50.0 mL of deionized water for 24.0 h at 37.0°C, then, the surface moisture was removed with filter paper, and the samples were weighed. Three sets of parallel values were measured, and the average value was calculated as the swelling degree of the sample. The sample swelling degree is calculated by equation (2):
where S is the swelling degree (%), M is the initial mass (g), and MS is the swelling mass (g).
2.7.5 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.
2.7.6 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 films were placed in a torsion fastness tester (XK-3014, Jiangsu Xiangke Instrument Company, China). The film 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× magnification.