3D Printing of PLLA/Biomineral Composite Bone Tissue Engineering Scaffolds

Tissue engineering is one of the most effective ways to treat bone defects in recent years. However, current highly active bone tissue engineering (BTE) scaffolds are mainly based on the addition of active biological components (such as growth factors) to promote bone repair. High cost, easy inactivation and complex regulatory requirements greatly limit their practical applications. In addition, conventional fabrication methods make it difficult to meet the needs of personalized customization for the macroscopic and internal structure of tissue engineering scaffolds. Herein, this paper proposes to select five natural biominerals (eggshell, pearl, turtle shell, degelatinated deer antler and cuttlebone) with widely available sources, low price and potential osteo-inductive activity as functional particles. Subsequently compounding them into L-polylactic acid (PLLA) biomaterial ink to further explore 3D printing processes of the composite scaffold, and reveal their potential as biomimetic 3D scaffolds for bone tissue repair. The research results of this project provide a new idea for the construction of a 3D scaffold with growth-factor-free biomimetic structure, personalized customization ability and osteo-inductive activity.


Introduction
Bone plays an indispensable role in the human body, and it is required for mechanical support in many human movements. Therefore, either damage or loss of bone tissue can cause great inconvenience to patients, and the treatment of large-area bone defects still faces great challenges. Autologous bone transplantation is the "gold standard" in the field of bone repair, but it is difficult to achieve because of secondary injury to patients [1,2]. Allogeneic bone transplantation also has many deficiencies, such as immune rejection and infectious diseases [3]. Tissue engineering aims to maintain, restore and enhance the function of injured or diseased tissues and organs by developing substitutes [4][5][6]. In recent years, significant progress has been made in tissue engineering, such as the discovery of methods to generate induced pluripotent stem cells [7,8], the finding that substrate stiffness can modulate stem cell differentiation [9], refined delivery mechanisms that enable biochemical cues such as growth factors and cytokines to be presented with improved bioavailability and bioactivity [10,11], increased understanding of the interaction between foreign bodies and the body's immune surveillance system [12,13], the development of new biomaterials and scaffolds that has led to fabrication of better biomimetic tissues [14,15],

Preparation and Characterization of Powder
The 5 kinds of biominerals, eggshell, pearl, turtle shell, degelatinated deer antler and cuttlebone were bought from Beijing Tongrentang. The biominerals were washed clean using deionized water and placed inside a fume hood to dry. A powder mill (Beijing Kewei Yongxing Instrument Co., Ltd., Beijing, China) was used to grind the 5 kinds of biominerals into powder, then a 50 μm sieve was used to screen, and finally collect and was set aside. The 5 kinds of biomineral powder were taken and handed over to the Material Center Laboratory of Tsinghua University for particle size, XRD and FTIR testing. The XRD values were matched with JCPDS values to confirm the mineral composition of the powders. XRD test results were analyzed using MDI jade 6 (Materials Data, Livermore, CA, USA), Omnic V8.2 (Nicolet, Madison, WI, USA) was used to analyze the FTIR test results, and OriginPro 8.5 (OriginLab, Northampton, MA, USA) was used for image rendering.

Preparation and Characterization of Ink
Take 0.15 g eggshell powder, pearl powder, turtle shell powder, degelatinated deer antler powder and cuttlebone powder, respectively, and put them into 10 mL 1,4-dioxane (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and use a cell ultrasonic crusher (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) for 5 min to disperse the powder in 1,4-dioxane. Add 1.5 g PLLA (viscosity 2.4, Beijing Jude Antai Technology Co., Ltd., Beijing, China) and stir at a constant temperature of 40 °C until PLLA is completely dissolved and the powder is evenly dispersed. Pour the prepared ink into the barrel of the 3D printer for standby. Use a 3D printer to print several inks under the pressure of 20, 40, 60, 80, 100, 120, 140 and 160 kPa, respectively, that is, draw a circle with a diameter of 10 mm, and then test the viscosity of the ink and select the appropriate printing pressure by observing its forming effect. The 3D image is drawn by the 3D builder, and the size of the circle is 10 mm × 10 mm × 0.5 mm. A stainless-steel plate was used as the receiver stage for the materials. The inner diameter of the 3D printer nozzle is 410 μm. The temperature is −3 °C.

Preparation of Scaffolds
Draw a 10 mm × 10 mm × 2 mm cuboid 3D image with the 3D builder and import it into the 3D printer control program (Gesim, Radberg, Germany). The 3D printing method

Preparation and Characterization of Powder
The 5 kinds of biominerals, eggshell, pearl, turtle shell, degelatinated deer antler and cuttlebone were bought from Beijing Tongrentang. The biominerals were washed clean using deionized water and placed inside a fume hood to dry. A powder mill (Beijing Kewei Yongxing Instrument Co., Ltd., Beijing, China) was used to grind the 5 kinds of biominerals into powder, then a 50 µm sieve was used to screen, and finally collect and was set aside. The 5 kinds of biomineral powder were taken and handed over to the Material Center Laboratory of Tsinghua University for particle size, XRD and FTIR testing. The XRD values were matched with JCPDS values to confirm the mineral composition of the powders. XRD test results were analyzed using MDI jade 6 (Materials Data, Livermore, CA, USA), Omnic V8.2 (Nicolet, Madison, WI, USA) was used to analyze the FTIR test results, and OriginPro 8.5 (OriginLab, Northampton, MA, USA) was used for image rendering.

Preparation and Characterization of Ink
Take 0.15 g eggshell powder, pearl powder, turtle shell powder, degelatinated deer antler powder and cuttlebone powder, respectively, and put them into 10 mL 1,4-dioxane (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and use a cell ultrasonic crusher (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) for 5 min to disperse the powder in 1,4-dioxane. Add 1.5 g PLLA (viscosity 2.4, Beijing Jude Antai Technology Co., Ltd., Beijing, China) and stir at a constant temperature of 40 • C until PLLA is completely dissolved and the powder is evenly dispersed. Pour the prepared ink into the barrel of the 3D printer for standby. Use a 3D printer to print several inks under the pressure of 20, 40, 60, 80, 100, 120, 140 and 160 kPa, respectively, that is, draw a circle with a diameter of 10 mm, and then test the viscosity of the ink and select the appropriate printing pressure by observing its forming effect. The 3D image is drawn by the 3D builder, and the size of the circle is 10 mm × 10 mm × 0.5 mm. A stainless-steel plate was used as the receiver stage for the materials. The inner diameter of the 3D printer nozzle is 410 µm. The temperature is −3 • C.

Preparation of Scaffolds
Draw a 10 mm × 10 mm × 2 mm cuboid 3D image with the 3D builder and import it into the 3D printer control program (Gesim, Radberg, Germany). The 3D printing method of normal temperature extrusion (nozzle diameter 410 µm) and low temperature (−3 • C) reception is used for printing. After printing, put the scaffolds into the freeze dryer for freeze-drying and finalize the shape. After 30 min of low-temperature vacuum drying, the shape of the scaffolds has been basically fixed, but it takes more than 2 h to completely remove 1,4-dioxane. In order to ensure the safety of the scaffolds, place in the fume hood for 7 days.

SEM Observation
The 5 kinds of biomineral powders were individually pasted onto the experimental bench and observed using a scanning electron microscope (S-450 SEM, Hitachi, Tokyo, Japan) after gold spraying. The SEM observation of the scaffolds is similar to this. The prepared scaffolds are cut into small pieces and pasted on the test bench. After spraying gold, the scaffolds can be observed by SEM. The SEM images of scaffolds were analyzed by Image J software, and the information regarding fiber diameter and aperture of different scaffolds was counted.

Cell Live/Dead Staining
Scaffolds of 10 mm × 10 mm × 2 mm in size were placed in a 24-well plate, and pure PLLA was set as control. SD-MSC P5 (purchased from Cyagen Biosciences Inc., Santa Clara, CA, USA) were seeded into the wells at a density of 5 × 10 4 cells in 500 µL medium per well. After the cells adhered to the wall for 4-6 h, scaffolds were moved into a new 96-well plate. After another 24 h, the medium was aspirated off and washed three times with PBS. Protected from light, calcein/propidium iodide working solution was formulated and replaced the previous medium, and was then incubated at 37 • C incubator for 15 min. The excessive dye was washed twice with PBS, and the well plate was moved to a confocal microscope (Zeiss LSM710, Oberkochen, Germany) for observation. Excitation wavelengths for PI and C-AM are 561 nm and 488 nm, and emission wavelengths for PI and C-AM are 579 nm and 537 nm, respectively. Scanning objective lens of 10× was used in the examination.

Cell Proliferation Assay
Briefly, 6 types of scaffolds were sterilized by 60 Co irradiation at 5 Mrad for further use. These scaffolds are cut to 5 mm × 5 mm × 2 mm in size and placed in 96-well plate, n = 3. SD-MSC P5 was resuspended in complete medium and evenly planted into 96-well plate at the density of 8000 cells in 200 µL medium per well. On 1/3/5 days, 10% v/v CCK8 working solution was prepared and replaced the previous medium. After incubation in 5% CO 2 incubator at 37 • C for 2 h, 100 µL suspension per well was moved into a new 96-well plate and absorption was measured by microplate reader at 450 nm. The quantitative data were plotted by Graphpad Prism V7 (GraphPad Software, San Diego, CA, USA) and statistically analyzed by SPSS 19 (IBM, Chicago, IL, USA).

ALP Detection
The preparation of cells and scaffolds is the same as that in 2.7. SD-MSC was seeded to the surface of the scaffold in the 24-well plate at the density of 5 × 10 4 cells in 500 uL medium per well. After 6 h of cell adhesion, the previous medium was replaced with osteogenic induction medium containing mem-α, 15% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 100 µM/mL of ascorbic acid, 2 mM of βglycerophosphate, 1.8 mM of KH 2 PO 4 and 10 nM of dexamethasone. The protein was extracted after 4/6 days of induction, and the protein concentration was measured at 595 nm by Bradford assay (semefeld article number: 23,200, standard curve is available on the official website). ALP concentration was measured at 405 nm according to the manufacturer's protocol for ALP Activity Kit (Sigma Aldrich, St. Louis, MO, USA). ALP activity = ALP concentration/total protein concentration.

Powder Test
To gain more detailed insight into the structure and composition of these five biominerals, the particle size, SEM, XRD, and FTIR tests were performed after the preparation for the five kinds of powders had been completed. The particle size distribution test results are shown in Figure 2.

Powder Test
To gain more detailed insight into the structure and composition of these five biominerals, the particle size, SEM, XRD, and FTIR tests were performed after the preparation for the five kinds of powders had been completed. The particle size distribution test results are shown in Figure 2. Most particle sizes are between 500-900 nm, while the diameter of the 3D printer nozzle is 410 μm, so particles of this size will not block the printer nozzle. However, some large particles are small in number but large in size, which will lead to the risk of nozzle blockage. Therefore, it is necessary to observe the powder via SEM under different magnification. As shown in Figure 3, from the SEM images with different magnifications, it can be seen that the particle size distribution range of the five kinds of biomineral powders is wide, ranging from nano scale to micron scale. Many larger sized particles are present in the powder, but they are all below 300 μm in size, posing a lesser risk of clogging of the nozzle. The observed particle sizes of these five kinds of biomineral powders were generally consistent and did not show large differences. In addition, it can also be seen in the SEM image that there is a certain phenomenon of mutual adsorption between particles, which will cause some trouble for 3D printing. Therefore, it should be fully dispersed in the ink preparation process. Most particle sizes are between 500-900 nm, while the diameter of the 3D printer nozzle is 410 µm, so particles of this size will not block the printer nozzle. However, some large particles are small in number but large in size, which will lead to the risk of nozzle blockage. Therefore, it is necessary to observe the powder via SEM under different magnification. As shown in Figure 3, from the SEM images with different magnifications, it can be seen that the particle size distribution range of the five kinds of biomineral powders is wide, ranging from nano scale to micron scale. Many larger sized particles are present in the powder, but they are all below 300 µm in size, posing a lesser risk of clogging of the nozzle. The observed particle sizes of these five kinds of biomineral powders were generally consistent and did not show large differences. In addition, it can also be seen in the SEM image that there is a certain phenomenon of mutual adsorption between particles, which will cause some trouble for 3D printing. Therefore, it should be fully dispersed in the ink preparation process. The mineral composition of these five kinds of biominerals can be determined by XRD ( Figure 4) and FTIR ( Figure 5).  The mineral composition of these five kinds of biominerals can be determined by XRD ( Figure 4) and FTIR ( Figure 5). The mineral composition of these five kinds of biominerals can be determined by XRD ( Figure 4) and FTIR ( Figure 5).  According to the analysis results of XRD and FTIR, the main chemical component of minerals in eggshell, pearl and cuttlebone is CaCO3, while the main chemical component of minerals in turtle shell and degelatinated deer antler is Ca5(PO4)3(OH). Among them, the main mineral type in eggshell is calcite; pearls contain two crystal types of CaCO3, aragonite and vaterite; the main mineral type in cuttlebone is aragonite; and the main mineral type in turtle shell and degelatinated deer antler is hydroxylapatite. Therefore, through the comparison of these five kinds of biominerals, whether there are differences in the application of biominerals containing different mineral types in the field of BTE can According to the analysis results of XRD and FTIR, the main chemical component of minerals in eggshell, pearl and cuttlebone is CaCO 3 , while the main chemical component of minerals in turtle shell and degelatinated deer antler is Ca 5 (PO 4 ) 3 (OH). Among them, the main mineral type in eggshell is calcite; pearls contain two crystal types of CaCO 3 , aragonite and vaterite; the main mineral type in cuttlebone is aragonite; and the main mineral type in turtle shell and degelatinated deer antler is hydroxylapatite. Therefore, through the comparison of these five kinds of biominerals, whether there are differences in the application of biominerals containing different mineral types in the field of BTE can be judged. Chemical composition and particle size of the biominerals are shown in Table 1.

Ink Test
In the process of 3D printing, the ink is extruded by the nozzle under appropriate air pressure and adhered to the receiving platform or the previous layer of material, and quickly frozen in a low temperature environment. Therefore, the selection of appropriate air pressure plays a vital role in 3D printing. Air pressure that is too low will lead to problems such as difficult extrusion of ink, and air pressure that is too high will lead to problems such as overly thick fiber, deformation and slowing down the speed of freezing. As shown in Figure 6, the printing effects of six kinds of composite inks (pure PLLA, PLLA/eggshell powder, PLLA/pearl powder, PLLA/turtle shell powder, PLLA/degelatinated deer antler powder and PLLA/cuttlebone powder) at 20, 40, 60, 80, 100, 120, 140 and 160 kPa, respectively. Ideally, the 3D printed circle should have a diameter of 1 cm and a fiber width of 410 µm, as indicated by the line segments in the figure. Obviously, with the increase in air pressure, the width of the line gradually increases. Through the comparison of design effects and actual effects, it is possible to verify the printability of this material and find out the suitable air pressure. It can be seen from the figure that the best printing pressure of pure PLLA ink is 100-120 kPa, the best printing pressure of PLLA/eggshell powder composite ink is about 100 kPa, the best printing pressure of PLLA/pearl powder composite ink is 100-120 kPa, the best printing pressure of PLLA/turtle shell powder composite ink is about 120 kPa, and the best printing pressure of PLLA/degelatinated deer antler powder and PLLA/cuttlebone powder composite ink is 100-120 kPa. In a word, the addition of these five kinds of powders has little effect on ink viscosity. It is more appropriate to select the air pressure of 100 ± 20 kPa according to the actual situation in the printing process.
Furthermore, according to the size distribution of particles, it can be judged that nozzles with an inner diameter above 300 µm are not easily clogged and nozzles with an inner diameter below 300 µm may also be used, but the risk of clogging will increase and lead to a decrease in the success rate of scaffold preparation. Nozzle clogging with a larger inner diameter carries little risk, but the increased diameter slows the freezing rate which in turn causes deformation of the scaffolds. Reducing the ambient temperature can accelerate the rate of freeze forming and reduce the risk of deformation. If the receiving base plate is used to cool down rather than the overall environment, the temperature will increase as it builds away from the base plate. For the macro size of the support, when the base plate temperature is −3 • C, the scaffolds can be printed to about 4 mm at most, and the length and width of the scaffolds are determined by the size of the receiving plate. If the whole printing environment is cooled down, the maximum size is determined by the size of the machine's printing space. The ink mixed with biomineral powders is more likely to be cryo-shaped during printing, possibly related to the presence of powders.
PLLA/eggshell powder composite ink is about 100 kPa, the best printing pressure of PLLA/pearl powder composite ink is 100-120 kPa, the best printing pressure of PLLA/turtle shell powder composite ink is about 120 kPa, and the best printing pressure of PLLA/degelatinated deer antler powder and PLLA/cuttlebone powder composite ink is 100-120 kPa. In a word, the addition of these five kinds of powders has little effect on ink viscosity. It is more appropriate to select the air pressure of 100 ± 20 kPa according to the actual situation in the printing process. Furthermore, according to the size distribution of particles, it can be judged that nozzles with an inner diameter above 300 μm are not easily clogged and nozzles with an inner diameter below 300 μm may also be used, but the risk of clogging will increase and lead to a decrease in the success rate of scaffold preparation. Nozzle clogging with a larger inner diameter carries little risk, but the increased diameter slows the freezing rate which in turn causes deformation of the scaffolds. Reducing the ambient temperature can accelerate the rate of freeze forming and reduce the risk of deformation. If the receiving base plate is used to cool down rather than the overall environment, the temperature will increase as it builds away from the base plate. For the macro size of the support, when the base plate temperature is −3 °C, the scaffolds can be printed to about 4 mm at most, and the length and width of the scaffolds are determined by the size of the receiving plate. If the whole printing environment is cooled down, the maximum size is determined by the size of the machine's printing space. The ink mixed with biomineral powders is more likely to be cryo-shaped during printing, possibly related to the presence of powders.

SEM Observation of the Scaffolds
SEM observation was carried out on the prepared 3D printing porous scaffolds, as shown in Figure 7.

SEM Observation of the Scaffolds
SEM observation was carried out on the prepared 3D printing porous scaffolds, as shown in Figure 7. The scaffolds mixed with various biomineral powders have almost the same pore structure as pure PLLA scaffolds, that is, they have both micron and nano pores. This pore structure is one of the advantages of 3D printing porous scaffolds. As shown in the figure, the micro pores of the scaffolds can reach about 400 μm, which is conducive to cell migration, while the nano pores distributed on the surface of the scaffold increase the surface area of the scaffold, which is conducive to cell adhesion and growth. It can be seen in the cross-sectional view that nano pores fill the whole scaffold, and the morphology of nano pores distributed in the scaffold is almost the same as that on the surface of the scaffold. The scaffolds mixed with various biomineral powders have almost the same pore structure as pure PLLA scaffolds, that is, they have both micron and nano pores. This pore structure is one of the advantages of 3D printing porous scaffolds. As shown in the figure, the micro pores of the scaffolds can reach about 400 µm, which is conducive to cell migration, while the nano pores distributed on the surface of the scaffold increase the surface area of the scaffold, which is conducive to cell adhesion and growth. It can be seen in the cross-sectional view that nano pores fill the whole scaffold, and the morphology of nano pores distributed in the scaffold is almost the same as that on the surface of the scaffold. The 3D printing process conducted at low temperature is affected by many factors, which have a great impact on its printing effect, and it is very difficult to control its diameter stability. Image J was used to analyze the aperture ( Figure 8) and fiber diameter (Figure 9) of the SEM images of the scaffolds. In contrast, the scaffolds mixed with biomineral powder showed certain shrinkages, and the fiber diameter was slightly smaller than that of the control group. On the whole, the difference in the diameter of each group of fibers is mostly less than 100 µm, which is already an ideal state for scaffolds printed by the low-temperature condensation method.  Research on the use of biomineral powders for 3D printing after mixing them with polymeric materials has been extensive, and the means of 3D printing used are diverse. Scaffolds printed using the high-temperature melt method tend to have better molding results with straight fibers, smooth surfaces and fewer pores, and the actual effect is more similar to the design effect [35]. The scaffolds fabricated by the printing method used in  Research on the use of biomineral powders for 3D printing after mixing them with polymeric materials has been extensive, and the means of 3D printing used are diverse. Scaffolds printed using the high-temperature melt method tend to have better molding results with straight fibers, smooth surfaces and fewer pores, and the actual effect is more similar to the design effect [35]. The scaffolds fabricated by the printing method used in this paper were less effective in molding than those fabricated by the high-temperature Research on the use of biomineral powders for 3D printing after mixing them with polymeric materials has been extensive, and the means of 3D printing used are diverse.
Scaffolds printed using the high-temperature melt method tend to have better molding results with straight fibers, smooth surfaces and fewer pores, and the actual effect is more similar to the design effect [35]. The scaffolds fabricated by the printing method used in this paper were less effective in molding than those fabricated by the high-temperature melting method, but the pores formed by lyophilization made the surface of scaffolds rough and the pore structure was more conducive to cell adhesion and growth, it also had a better promotion effect on bone regeneration. The scaffolds mixed with biomineral powder did not show much difference in printability, scaffold morphology or pore structure compared to PLLA.

Cell Experiment
As shown in Figure 10, after 24 h of cell adhesion, the cells on the surface of the well plate are evenly distributed. The fluorescence test results showed that, in the sustainedrelease solution environment of each group of scaffolds, the cells were typical green living cells, while the red dead cells were basically invisible. The observation of cells on scaffolds in each group showed that the number of cells was relatively small, but most of them were still living cells. Most of these cells are distributed along the axial direction of the intersecting scaffold fibers, and a small number of cells will also gather in the pores of the scaffold. PLLA scaffolds are hydrophobic, so less cells remain on the fibers in the early stage of adhesion, and the long-term effect of cell proliferation needs to be verified by CCK8 experiments. However, the staining results showed that the cell state was good enough to confirm the excellent biosafety of the scaffold. As we can see in Figure 10B,C, >90% of cells at scaffolds were live on all groups, which indicated the satisfying biosafety of our PLLA and PLLA/biomineral scaffolds. As shown in Figure 11, the cells on all groups of scaffolds showed a typical growth trend on the 1st/3rd/5th day after seeding. From the 1st day to the 3rd day, the OD values of the control group (pure PLLA), pearl group and cuttlebone group increased significantly (p < 0.05), while the eggshell group, turtle shell group and degelatinated deer antler group had very significant statistical significance (p < 0.001). From the 3rd day to the 5th day, the increase of OD value in the PLLA group was statistically significant (p < 0.05), while other groups showed very significant statistical significance (p < 0.001). In the pure PLLA, eggshell and pearl groups, the OD values on the first day were 0.011 ± 0.009, 0.007 ± 0.002 and 0.008 ± 0.008, respectively, which were lower than the 0.045 ± 0.003, 0.04 ± 0.007 and 0.047 ± 0.002 in the turtle shell group, degelatinated deer antler group and cuttlebone group.
On the 5th day, the PLLA group (0.4 ± 0.075) and eggshell group (0.415 ± 0.046) were still lower than the pearl group (0.82 ± 0.03), degelatinated deer antler group (0.668 ± 0.059) and cuttlebone group (0.753 ± 0.012). At the same time, the OD value of the turtle shell group also increased to 0.634 ± 0.026. From the 1st day to the 5th day, the OD values of the turtle shell group, degelatinated deer antler group and cuttlebone group remained relatively high. In order to evaluate the effect of drug loaded scaffolds on osteogenic differentiation, the early index of osteogenesis, ALP activity, was measured. As shown in Figure 12, the ALP activity of PLLA on the 4th day was set as the control. From the 4th day to the 6th day of induction, the ALP activity of each group increased, and the OD of the turtle shell group, degelatinated deer antler group and cuttlebone group increased significantly (p < 0.05). On the 4th day, the ALP activity of the pearl group, degelatinated deer antler group and cuttlebone group were higher than that of PLLA group, eggshell group and turtle shell group. However, on the 6th day, the eggshell group and turtle shell group increased rapidly, which was higher than that of the pearl group. On the 4th and 6th day, the cuttlebone group was always the group with higher ALP activity. The stronger trends of osteogenic differentiation indicated higher potential of bone matrix secretion after implantation in vivo. After the cells were seeded on the scaffold, cells adhesion for 4~6 h in the first 24 h was crucial to their viability, so live/dead staining was performed on the first day. Due to its hydrophobicity, PLLA was usually queried for its bioactivity of osteogenic properties, such as cell migration, adhesion, proliferation and differentiation [44][45][46][47]. It was reported to be applicable as osteo-conductive but not osteo-inductive material [48,49]. Various modifications have been used for better cell behaviors on PLLA scaffolds, such as microscopic shaping, electrospinning, molding, and 3D printing [46,47,50,51]. Another direct functionalization is introducing other bioactive materials such as collagen, biominerals and growth factors [52][53][54]. Considering the stability and economic cost of involved materials, biominerals were applied here for better osteogenic induction and cell behaviors. Eggshell and pearls are common bioactive materials, but they only provide osteo-conductive function rather than osteo-inductive [55]. However, turtle shell, degelatinated deer antler and cuttlebone are traditional biomaterials [56][57][58][59]. Among the scaffold preparation methods, 3D printing is a promising strategy for bioactivity involvement, as well as strong customizability according to the specific morphology of in situ defect areas [60]. Similarly with previous publications, cells poorly adhered on PLLA scaffolds, but stayed well on the PLLA/biominerals scaffolds. The significantly improved ALP activities of SD-MSC showed exciting potential for bone matrix secretion and in vivo implantation. The results provide great evidence for further in vivo study, which would do great favor for bone defect repair in translational medicine. Some studies have shown that the organic matrix contained in biological minerals can promote the differentiation of bone marrow mesenchymal stem cells, but cell proliferation and differentiation have no obvious relationship with the molecular weight of organic matrices. For example, YS Liu [33,36] divided a pearl water soluble organic matrix into an organic matrix < 1 kDa and organic matrix > 3.5 kDa according to molecular weight, and compared the effects of an organic matrix with different molecular weights of aragonite and spheroidal aragonite on mouse MC3T3-E1 cells and human bone marrow mesenchymal stem cells. The results showed that the water-soluble organic matrix had no promoting effect on the proliferation of the two types of cells, but was able to promote the differentiation of the two types of cells into an osteogenic direction. Both the composition and structure of natural biominerals are more complex than those of artificial materials, so it is necessary in future studies to decompose their constituents to investigate the mechanism of action of their different constituents on cells, which will help to distinguish and refine more efficient fractions in biominerals to better promote the application of biominerals in the field of tissue engineering. At present, the mechanisms and applications of biominerals in the field of bone tissue engineering have been widely studied, and many other biominerals, except for the five kinds of biominerals studied in this paper, have been intensively studied, such as biosilica isolated from marine sponges et al. [61][62][63][64]. Studies have proven that biosilica isolated from marine sponges can function to balance pH, control calcium absorption/release and enhance cell viability/proliferation [65,66]. Similar to that, a large number of biominerals have now been shown to have the ability to promote cell proliferation and differentiation, which holds great promise in tissue engineering. In future studies, it is necessary to deeply investigate and contrast the commonalities and differences of biominerals on cell proliferation and differentiation and explore their effects under different usage scenarios to better advance their applications in the field of tissue engineering.

Conclusions
The main components of eggshell, pearl, turtle shell, degelatinated deer antler and cuttlebone are organic matter and calcium carbonate and/or calcium phosphate. These five kinds of biominerals all enable the fabrication of 3D porous scaffolds through the process of grinding, screening, compounding with PLLA to prepare 3D printing ink, and subsequent 3D printing and freeze-drying. These five kinds of 3D composite scaffolds all simultaneously possess macro and microscopic multi-stage pores, which are beneficial to cell migration, growth and adhesion. Cell experiments demonstrated the five biomineral composite scaffolds have good biocompatibility and the ability to promote bone regeneration. Therefore, biominerals have a promising future in the design and application of scaffolds for BTE.
Author Contributions: Conceptualization, F.G., W.Y. and X.S.; methodology, C.M. and W.W.; investigation, Y.X. and C.L.; writing-review and editing, F.G., W.Y. and C.M.; project administration, F.G. and X.S.; funding acquisition, F.G. All authors have read and agreed to the published version of the manuscript.