In vivo study of dual functionalized mussel-derived bioactive peptides promoting 3D-printed porous Ti6Al4V scaffolds for repair of rabbit femoral defects

Abstract

The 3D printed porous titanium alloy scaffolds are beneficial to enhance angiogenesis, osteoblast adhesion, and promote osseointegration. However, titanium alloys are biologically inert, which makes the bond between the implant and bone tissue weak and prone to loosening. Inspired by the natural biological marine mussels, we designed four-claw-shaped mussel-derived bioactive peptides for the decoration of porous titanium alloy scaffolds: adhesion peptide-DOPA, anchoring peptide-RGD and osteogenic-inducing peptide-BMP-2. And the bifunctionalization of 3D-printed porous titanium alloy scaffolds was evaluated in vivo in a rabbit model of bone defect with excellent promotion of osseointegration and mechanical stability. Our results show that the in vivo osseointegration ability of the modified 3D printed porous titanium alloy test piece is significantly improved, and the bifunctional polypeptide coating group E has the strongest osseointegration ability. In conclusion, our experimental design partially solves the problems of stress shielding effect and biological inertness, and provides a convenient and feasible method for the clinical application of titanium alloy implants in biomedical implant materials.

Keywords

3D printing porous titanium alloy dihydroxyphenylalanine RGD BMP-2 osseointegration

Introduction

Medical titanium alloy Ti6Al4V has been widely used in orthopedic surgery including total joint replacement, bone defect repair and spinal fusion.¹ Ti6Al4V has excellent biocompatibility, high corrosion resistance and hypoallergenicity.² Compared with other metals, titanium alloy is light in weight, low in elastic modulus, non-magnetic, low in thermal expansion coefficient, yield strength and fatigue strength, and has semi-transmissive to X-rays.³ However, Ti6Al4V still has obvious defects. First, the elastic modulus of Ti6Al4V is still relatively high for bone tissue, which is prone to "stress shielding effect", resulting in degeneration and absorption of surrounding bone tissue, which seriously affects the long-term effective use of the implant.⁴ On the other hand, titanium and titanium alloys are biologically inert materials, lacking biological activity, and it is difficult to directly form chemical bonds with bone tissue after implantation in the body.⁵ Therefore, the bonding strength of the prosthesis and the bone tissue is low, and it is easy to loosen.

In order to make up for the defects of titanium alloy materials, in view of the stress shielding phenomenon, the porous structure has entered the design idea of metal materials for bone tissue repair. Recently, an emerging metal rapid prototyping technology, Electron Beam Melting (EBM), can realize the fine control of pore parameters and the preparation of porous metal materials with complex shapes.⁶ The previous research of the research group showed that different pore size or porosity not only determines that the mechanical properties of the material can match the mechanical properties of the host bone tissue,⁷ but also the rough inner and outer surfaces of the porous structure also affect the bone formation at the bone-implant interface.^8,9 When designing the scaffold, the Ti6Al4V scaffold with a pore size of 600 μm was selected, which not only has sufficient mechanical strength, but also promotes bone cell adhesion, proliferation and early differentiation at the same time.¹⁰ On the other hand, biologically inert titanium alloys can only rely on the self-healing ability of the tissue organism if implantation is successful.¹¹ Titanium alloy surfaces lack biological activity to induce osteocytes to form tissue responses (eg, adhesion, signaling, and stimulation) at the bone-implant interface.¹² Therefore, it is important to find specific bioactive substances that can induce cell adhesion and accelerate bone regeneration at the bone-implant interface to reduce the risk of implant loosening.^13,14

In the context of biomimicry, marine mussels have received significant attention. They attach themselves to wet rocks with natural adhesives on their feet.¹⁵ One such binder is 3,4-dihydroxyphenylalanine (DOPA), whose catechol group readily reacts with the substrate in a covalent manner in aqueous solution.¹⁶ Studies have shown that,¹⁷ the surface of titanium implants sealed in air has a tight and continuous layer of titanium oxide (TiO2). The catechol moiety of DOPA was adsorbed onto the Ti surface by means of bidentate chelation with TiO molecules, which enhanced the adhesion. Therefore, rational design of catechol-containing biomolecules to improve cell adhesion and osteogenesis has great clinical application value for bone regeneration on the surface of porous titanium alloy implants.

Arginine-Glycine-Aspartic (RGD) polypeptides were originally identified as sequences within fibronectin that mediate cell attachment.¹⁸ RGD is a cell recognition and attachment site for many extracellular matrix proteins and cell surface proteins involved in the regulation of cell adhesion, cell spreading, and actin cytoskeleton formation.¹⁹ In fact, the synthesis of RGD peptides is relatively simple and inexpensive, and RGD-peptides are widely used in tissue engineering by chemically modifying peptides, liposomes, and polymers.^20,21 It has been reported in the literature that immobilization of RGD peptide on titanium surface can promote bone tissue regeneration by promoting early osteoblast attachment and subsequent proliferation and differentiation functions, thereby enhancing implant bone healing.^21,22

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor beta (TGF-β) superfamily,²³ about 20 BMP isoforms have been identified.²⁴ Although many BMPs have osteogenic properties, BMP-2 is currently the strongest osteogenic induction factor involved in regulating cell proliferation, differentiation, apoptosis, and physiological activities such as cartilage induction, bone formation, and organ formation.²⁵ Therefore, BMP-2 is the first and only one to be introduced as a bone graft substitute.²⁶ However, the short half-life of BMP-2 has the disadvantage of too fast release, which greatly limits the application of BMP-2.²⁷ The new BMP-2 peptide synthesized by our research group using the core sequence of BMP-2 protein (KIPKASSVPTELSAISTLYL) as raw material can maintain the characteristics of long-term activity and overcome the decline in biological activity caused by the spatial conformation of the natural protein shielding some sites.²⁸

To sum up, in order to better clinical application of porous titanium alloy stent, it is the core purpose of this study to find an ideal bioactive substance to form a stable biological connection between the implant and the surrounding bone tissue. Combined with previous experimental content,^29,30 in this study, porous titanium alloys were prepared by 3D printing, which reduced the elastic modulus of the materials, increased the ingrowth of bone tissue, and used mussel-like biomimetic peptides to modify the surface of the material to improve the "inertness" of the titanium alloy surface. In this study, we innovatively designed Dopa peptide, RGD peptide and BMP-2 peptide, and applied dual biofunctional active peptides to promote early osseointegration of porous titanium alloy implant-bone interface in New Zealand rabbits and improve biomechanical stability sex. We expect that the dual bioactive peptides demonstrated in this study for porous titanium alloy implants will help improve the application of medical implants and provide new theoretical research value for clinical use.

Materials and methods

Experimental animals

30 healthy male New Zealand white rabbits aged 3 months, ordinary grade, weighing 2.0–2.2 kg, purchased from the Animal Experiment Center of Soochow University, license number: SYXK (Su) 2014–0029, certificate number: 201829792, free access to food and water. This animal experiment was completed at the Institute of Orthopedics, Soochow University from February 2020 to May 2020. All experimental animals were provided by the Laboratory Animal Center of Soochow University, approved by the Animal Management Committee of Soochow University, and animal experiments were carried out in strict accordance with the guidelines of the "Protection and Use of Laboratory Animals" issued by the China Animal Research and Animal Management Committee.

Reagents

Three mussel-like biomimetic peptides: DOPA peptide, RGD peptide, BMP-2 peptide were designed by the research group and produced by Shanghai Qiangyao Biotechnology Co., Ltd. Phosphate-buffered saline solution (PBS, 0.02 mmol/L, pH = 7.2) prepared with Milli-Q water, purified using the Thermo Scientific Barnstead NANOpure Diamond Water Purification System with a resistivity of at least 18.2 MΩ•cm and purchased phosphate-buffered saline (Beyotime Biotechnology, China). The following reagents are purchased directly: 75% ethanol, An'er iodine (Shanghai Zhongyou Pharmaceutical Co., Ltd., China); Dibutyl phthalate (Tianjin Fuyu Fine Chemical Co., Ltd., China); Toluidine blue stain (Nanjing Jiancheng Bioengineering Institute, China); Sodium pentobarbital (Sigma, USA); Penicillin for injection (Beijing Zhongshan Golden Bridge Co., Ltd., China).

Instruments and equipment

Small animal digital X-ray imaging system (SedecalSPL-HF-VET-4.0, Spain); Small animal tomography system (SkyScan1176In-Vivo Micro-CT, Belgium); Optical microscope (ZEISS Image M1, Germany); Microcomputer-controlled electronic universal material testing machine (HY-1080 10000 N, China); Orthopedic surgical instruments (Zhangjiagang Golden Deer Medical Instrument Co., Ltd., China); 3D printed porous titanium alloy stent (Beijing Aikang Yicheng Medical Instrument Co., Ltd., China); High pressure steam sterilizer (Bajin Experimental Equipment Co., Ltd., China); Ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd., China); Constant temperature water bath (Shanghai Shuoguang Electronic Technology Co., Ltd., China); Hard tissue slicer (Leica, Germany); Flaker (Ruifeng Instrument Equipment Co., Ltd., China); Tension-torsion biaxial electronic universal material mechanical testing machine (Instron Corporation Norwood, USA); X-ray photoelectron spectroscopy (XPS, ESCALAB 250 XI, Thermo Fisher Scientific, USA); Scanning electron microscope (FE-SEM, Zeiss, Germany, SUPRA55).

Preparation of porous titanium alloy scaffolds

The CAD software Materialise/MagicsTM (Materialise Belgium) was used to design and establish a three-dimensional digital model of the porous titanium alloy implant rod. Its external shape is cylindrical, with a diameter of 6 mm and a height of 10 mm. about 600 μm. Then, the digital model is cut into several slices with a fixed layer thickness of 100 μm, and the data of each slice is obtained. The acquired slice data were imported into EBM equipment (ArcamAB, Sweden). Then, in a vacuum environment, at a temperature of 650°C, a thin layer of Ti6Al4V powder (particle diameter 45–100 μm) was preheated with an electron beam current of 30 mA, and the scanning speed was 15000 mm/s. Then, the Ti6Al4V powder raw material was melted layer by layer at a scanning speed of 6 mA and 400 mm/s according to the imported tomographic data under computer control, and finally a porous Ti6Al4V implanted rod with the shape and internal structure consistent with the design model was obtained (Figure 1).

Axial compression experiments were performed on porous titanium alloy stents (speed: 1 mm/min) using an electronic universal material testing machine, and three samples were measured. The deformation curves were plotted and the average elastic modulus yield strength of the porous titanium alloy scaffolds was calculated. The calculation formula of yield strength is σ = F/A, the unit is Mpa, σ is the yield strength, F is the force at the yield point, and A is the bottom area of the material. The calculation formula of the elastic modulus is E = σ/ε = Fh/LA, E is the elastic modulus, F is the test stress at a certain point on the line, h is the material height, L is the material deformation at a certain point on the line, and A is the material bottom area.

Design and synthesis of three mussel bioactive peptides (DOPA peptide, RGD peptide and BMP-2 peptide)

Inspired by mussel byssin proteins in marine mussels, the catechol in DOPA amino acids can have a strong coordination reaction with TiO2 on the surface of titanium alloys, so we designed a DOPA peptide containing 4 DOPA amino acids, the sequence of which is Ac-(DOPA)-G-(DOPA)-K(PEG5-Azido)-(DOPA)-G-(DOPA), AC is the protecting group, G is the spacer, and K(PEG5-Aziod) is responsible for interacting with RGD The group to which the peptide, BMP-2 peptide is linked. The other two biologically active peptides are RGD peptide, which plays a specific cell adhesion function, and BMP-2 peptide, which plays an osteoinductive function. The sequence of the RGD peptide is (DBCO-Mal)-CGGGGGRGDS, and the sequence of the BMP-2 peptide is (DBCO-Mal)-CGGGGKIPKASSVPTELSAISTLYL, GGGG is a spacer, used to increase the accessibility of biologically active peptides, (DBCO-Mal)-C is the group responsible for connecting with DOPA peptides, GRGDS sequence is an adhesive short peptide, play a role in The main activity is the RGD tripeptide sequence, which can promote the adhesion of osteoblasts to the surface of the material. KIPKASSVPTELSAISTLYL is the core sequence of BMP-2 protein, which can exert the same osteogenic activity as BMP-2 protein, and is more stable than BMP-2 protein. The design concept of mussel-like biomimetic peptides and the mechanism of surface modification to promote osseointegration are shown in Figure 2.

The three peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. with the help of Fmoc-DOPA(acetonide)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc -Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, DBCO-MalAzido-PEG5-CH2CH2COOH, acetic anhydride as raw material, after resin swelling, deprotection, washing, condensation, drying, cutting, purification, lyophilization The chemical structures of the two biomimetic peptides were confirmed by 1 H and 13 C NMR. wherein the Fmoc group is a protecting group. The lyophilized polypeptide was in the form of white powder. Finally, a small part of the three lyophilized bioactive peptides were taken out for molecular weight identification by MS and purity identification by HPLC analysis. After being identified as the target sequence, the lyophilized bioactive peptides were stored at -20°.

The porous titanium alloy stent was soaked in acetone overnight, placed in an ultrasonic cleaner for 5 min; soaked in acetone again for 15 min, and ultrasonically cleaned for 10 min, repeated three times; then, soaked in absolute ethanol for 15 min, and ultrasonically cleaned for 5 min, repeated three times; plasma cleaning for 5 min (Voltage 600–800V, flowmeter 20–30 mL/min), repeat 2 times. The cleaned porous titanium alloy stents were first sterilized by high temperature and high pressure, then soaked in DOPA peptide solution (0.1 mg/mL) in the dark for 24 h, and finally soaked in different ratios of RGD peptide (0.1 mg/mL) and BMP in the dark. -2 peptide solution (0.1 mg/mL) for 24 h, after soaking, the porous titanium alloy scaffold was taken out and rinsed three times with PBS. The modified porous titanium alloy scaffolds were set as PBS group (group A), DOPA peptide group (group B), DOPA peptide+RGD peptide group (group C), DOPA peptide+BMP-2 peptide group (group D), DOPA peptide+RGD peptide+BMP-2 peptide group (group E). Among them, group A was the control group soaked in PBS solution only, and the other groups were modified with different ratios of peptides. According to the previous research results of the research group on modified TiO2 similar peptides,^29,30 the ratio of DOPA peptide: RGD peptide: BMP-2 peptide in each group is: B peptide group (4:0:0), C peptide group (4:4:0)), D peptide group (4:0:4), E group: (4:1:3).

Scanning electron microscope to observe the surface morphology of porous titanium alloy implants

Scanning electron microscope (SEM) was used to observe whether the surface morphology of titanium alloy stents in each group changed. Because the height requirement of SEM for the sample cannot exceed 5 mm, and the observed surface must be flat, so in advance, a cylindrical titanium alloy bracket with a height of 10 mm is cut into two cylindrical brackets with a height of 5 mm, and the cylinder is observed with SEM. The uncut bottom surface, because the titanium alloy itself has conductivity, and only a thin layer of polypeptide covers the surface, so it can be observed directly under SEM without gold spraying.

X-ray photoelectron spectroscopy (XPS) to detect the chemical composition of porous titanium alloy surface coatings

The porous titanium alloy stents were soaked in the polypeptide solution for 24 h, dried with a hair dryer, and the samples were prepared. The characteristic peaks of various elements were measured on each group of porous titanium alloy stents with an X-ray photoelectron spectrometer (test tube voltage: 15kv, tube current: 10 mA). The detection, including the ratio of titanium (Ti), aluminum (AI), oxygen (O), nitrogen (N), vanadium (V) and other elements. At the same time, the proportion of various elements is calculated. To determine the repeatability of the coating surface chemistry, 3 measurements were performed on each sample.

Design of animal experiments

In this experiment, 30 New Zealand rabbits were randomly divided into 6 groups according to the random number table method, with 5 rabbits in each group. One group was the defect control group, and the other 5 groups were implanted porous titanium alloy test pieces. According to the different coatings of titanium alloy test pieces, it is divided into group A (untreated porous titanium alloy), group B (porous titanium alloy functionalized with Dopa peptide), group C (porous titanium alloy functionalized with Dopa peptide + RGD peptide) titanium alloy), group D (porous titanium alloy functionalized with Dopa peptide+BMP-2 peptide), group E (porous titanium alloy bifunctionalized with Dopa peptide+RGD peptide+BMP-2 peptide). Lateral X-ray films were taken for all New Zealand rabbits at 4 and 8 weeks after the animal modeling operation; all rabbits were killed by excessive sodium pentobarbital at 8 weeks after operation, and the complete femur specimens were taken out for gross observation; -CT detection of trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), trabecular bone relative volume (BV/TV) and trabecular bone separation (Tb.Sp); hard tissue section staining observation Osseointegration differences.

Femoral condyle implantation with porous titanium alloy Ti6Al4V scaffold

All surgical instruments were autoclaved (121°C, 15 min, 1.05 kg/cm²) before surgery. All porous titanium alloy stents were cleaned by ultrasonic vibration before implantation in experimental animals, and sterilized by high temperature and high pressure steam. The animals were anesthetized by injection of 2% sodium pentobarbital (30 mg/kg) through the ear vein before surgery. After the anesthesia took effect, the surgical area was prepared for skin, the rabbit was fixed on the operating table, and the surgical area was sterilized twice with Aner's iodine, and a towel was spread. A 2 cm incision was made on the lateral condyle of the femur of the rabbit. The skin, subcutaneous, fascia, and bones are incised in sequence, and the periosteum is partially peeled off. As shown in Figure 3, In the lateral center of the femoral condyle, a bone tunnel (diameter = 6 mm, length ≥10 mm) was opened with an osteotome perpendicular to the longitudinal axis of the femur. The wound was flushed with normal saline containing penicillin, and the porous titanium alloy scaffold was completely implanted into the bone tunnel. After confirming its position and stability, the incision was sutured layer by layer. Intramuscular injection of penicillin (160×10⁴ U/d) was administered within 3 days after the operation to prevent infection. And X-rays were performed on the day of surgery to check the implant position.

General observation after operation

The hair, diet, excretion and activities of the experimental animals were observed daily after the operation; before the surgical incision was restored, it was necessary to pay attention to whether there were signs of infection such as self-mutilation, redness, swelling, oozing and exudation. There were no deaths before the end of the experiment. Eight weeks after the operation, the rabbits were euthanized, and the femur specimens were taken out to observe the healing of bone defects in each group, whether there was surrounding tissue, fibrous tissue dissolution and necrosis, and to observe the formation of new bone on the implant surface.

X-ray film observation

At the fourth and eighth week after operation, the lateral X-ray films of the bilateral hindlimbs of the experimental animals were taken using the small animal digital X-ray imaging system to observe whether the implants were displaced, prolapsed, and whether there was osteolysis around the implants, osteonecrosis.

Micro-CT detection

Eight weeks after the operation, the condyle of the rabbit femur containing the titanium alloy scaffold was taken out, the soft tissue attached to the bone was removed, and the relevant information was marked, and then it was fixed in 80% ethanol for 2 weeks. Scan the sample with Micro-CT for 3D reconstruction. Relevant parameters: voltage 80 kV, current 500 μA, rotation angle: 360°, spatial resolution 14 μm, pixels 1336 × 2000, exposure time 290 ms. In the surface porous scaffold structure of the curved surface of each specimen, a rectangle with a length of 2.3 mm and a width of 1.3 mm was selected, and the 250th layer was the region of interest. The Micro-CT supporting software CT Analyser (Version: 1.16.4.1) was used for analysis, and the quantitative analysis of the images included bone volume fraction (bone volume/total volume, BV/TV, %), trabecular bone number (Tb N, 1/mm), mean trabecular bone thickness (Tb. Th, mm) and trabecular bone separation degree (Tb. Sp, mm).

Histomorphological analysis

Paraformaldehyde-fixed samples were dehydrated with graded (70–100 wt%) ethanol solutions and then embedded in a 37°C polymerized methyl methacrylate (MMA) solution. Thin sections (approximately 50 μm thick) were sawn and polished, notably, depending on the central position of the implant's longitudinal axis, cutting in a direction perpendicular to its longitudinal axis was required. Then stained with 1% toluidine blue staining solution. After mounting, an optical microscope was used to capture images to observe the formation of new bone tissue around the porous titanium alloy material and in the pores.

Statistical analysis

The sample size for each experiment is at least 3. All quantitative experimental data were statistically analyzed using SPSS 14.0 for Windows software (SPSS, Chicago, IL, USA). Statistical methods used one-way analysis of variance (ANOVA) and Tukey's multiple comparison test, experimental data were recorded as mean ± standard deviation (‾x ± S), defined as statistical significance when *p < 0.05, **p < 0.01 statistically significant.

Result

Mechanical properties of porous titanium alloy implants

As shown in Figure 4, it can be seen from the load-displacement curve that with the increasing load on the porous titanium alloy stent by the universal mechanical test machine, the deformation of the stent also increases, and the maximum bearing force before the stent fails is 4119.45 ± 268.57 N, the maximum deformation is 1.02 ± 0.08 mm, the yield strength is about 130.5 ± 15.5 Mpa, and the elastic modulus is about 2.2 ± 0.1 GPa.

The morphology of porous titanium alloy scaffolds was observed by SEM.

The surface morphologies of each group of different coatings on the surface of 3D printed titanium alloy Ti6Al4V are shown in Figure 5. The micropores of the porous titanium alloy scaffolds can be clearly seen in each group under a 30x microscope, with a pore size of about 600 μm. It can be seen that the entire surface of group A is covered with a layer of thin film with a gray lamellar structure, and the structure is complete and continuous without obvious fracture and overlap. In contrast, the surfaces of Groups B, C, D, and E can be clearly seen covered with material, which is manifested as a significant increase in the displayed roughness. Interestingly, the cross-sections of the porous structures on the titanium alloy scaffolds can be seen in groups D and E, which are considered to be left by the cutting process of the titanium alloy scaffolds after 3D printing. Under the microscope at 500°, a large number of spherical structures can be seen in group B, which are formed by incompletely molten Ti6Al4V powder when preparing porous titanium alloy scaffolds for EBM. Under the 30x and 500x SEM microscopes, no changes in the morphology of the porous titanium alloy scaffolds after surface modification were found. The results of scanning electron microscopy showed that the morphology of the titanium alloy scaffold did not change after the surface modification of the mussel-like biomimetic polypeptide.

The surface-functionalized titanium alloy scaffolds were characterized by X-ray photoelectron spectroscopy (XPS). As shown in the Figure 6, after treatment with DOPA bioactive peptide, the signal of N1s element can be detected. The N1s element signal of the surface-grafted RGD+BMP-2 dual-energy bioactive polypeptide E group was significantly enhanced. As expected, the quantitative results showed that the N element concentration in group B increased from 1.52% to 3.89% relative to group A (no peptide coating). The N element concentration of group E increased from 1.52% to 6.15%, and the increase was about 8.05%. Compared with group B, the concentration of N element in group C increased from 3.89% to 6.15%, with an increase of about 2.26%; the concentration of N element in group D increased from 3.89% to 8.52%, with an increase of about 4.63%.

General observation results of each group

Rabbits' movements and diet were normal except for the limp due to wound pain for 7 days after surgery. The incision recovered well after the operation, and no inflammation such as redness or exudation was found. At the eighth week after the operation, as shown in Figure 7, the bone around the defect was obviously sunken, the boundary of the bone defect was still clear, and the defect was filled with scars and completely repaired. In groups A, B, C, and D, the implants were closely connected with the surrounding bone tissue, the boundary of the bone defect was blurred, and there was no obvious dissolution or necrosis of the surrounding tissue. Among the 8 specimens in group E, the situation shown in the figure occurred in 3 specimens, and the situation in group E occurred in the rest of the groups. The material was covered by a large number of callus, and the surface of the material could not be directly observed.

As shown in Figure 8. Postoperative X-ray pictures showed that all implants were successfully implanted, the size perfectly matched the femoral condyle, no obvious displacement and detachment, and no macroscopic infection response. Lateral X-ray films of the rabbit's bilateral hind limbs were taken at 4 and 8 weeks, respectively. As shown in Figure 9, the X-ray films at 4 and 8 weeks after the operation showed that the implants in each group were all located in the femoral condyle, without displacement and loss, the material was well integrated with the bone, and no osteolysis and bone loss were observed. necrosis phenomenon. Among them, the low signal area around the experimental scaffold in group E was significantly smaller than that in the other groups, and the material was most closely combined with bone.

Micro-CT analysis of osseointegration

Eight weeks after surgery, the porous titanium alloy stent implanted in the experimental rabbit was taken out for Micro-CT detection. Assessing the osteogenesis around the implant (Figure 10), it can be seen that the gray fuzzy tissue in the porous structure of group E is significantly more than that of other groups, and that of groups B, C, and D is significantly more than that of group A. The 3D reconstructed image (Figure 11) created by the onboard software can observe that the new bone is marked in dark red and the titanium alloy Ti6Al4V is marked in white. It can be seen that the area covered by new bone on all coated titanium alloy stents is significantly more than that of group A, and the dark red color of group E is the most dense. According to the measurement data of the onboard software, the statistical analysis was carried out, and the results are shown in Figure 12: Compared with the other four groups, the bifunctional group E obviously showed the highest bone percentage (BV/TV), Similarly, the number of trabecular bone (Tb.N, 1/mm) and the average thickness of trabecular bone (Tb.Th, mm) were also the best bone condition in the bifunctional E group, and the results were statistically significant Scientific significance (p < 0.01). On the contrary, the degree of trabecular bone separation (Tb. Sp, mm) decreased to varying degrees (p < 0.01).

Histological and histomorphological examination

Next, to assess the interfacial bonding between the implant and native bone, we sectioned the hard tissue and stained it with toluene blue. As shown in the Figure 13, the implanted titanium alloy scaffold is shown in black, the fibrous tissue is stained light yellow, and the purple represents the new bone tissue. It can be observed that a large amount of fibrous tissue is formed inside group A, while gaps can still be seen on its surface with less new bone formation. In contrast, groups B, C, D, and bifunctional E showed the newly formed bone more clearly. We noticed that the bifunctional group E implants in all porous implants showed denser interfacial osseointegration strength and excellent bone implantation ability compared with other groups. Almost all the superficial micropores had new bone tissue ingrowth, and some of the new bone extended into the deep pores of the experimental scaffold, and tightly combined with the porous structure to form mechanical interlocking.³¹ In the single bioactive polypeptide group, only part of the surface micropores had new bone tissue ingrowth, and the new bone was poorly integrated with the implant. Furthermore, in the bifunctional E group, the periphery of the titanium alloy scaffold showed a continuous bone matrix and a significant increase in the surrounding trabecular bone. It is also worth mentioning that no obvious inflammatory mediators such as multinucleated foreign body giant cells or fibrous capsules were observed in the bone and implants of all groups.

Discuss

Bone tissue is known to be a dynamic tissue capable of repairing mild injuries through bone remodeling.³² However, for patients with severe bone defects, bone tissue cannot repair damage by itself.³³ Replacement of damaged bone tissue with bioactive biomaterials to assist tissue reconstruction is necessary.^34,35 Because orthopedic implants require a high degree of load-bearing,³⁶ the titanium alloy Ti6Al4V is widely used in biomedical equipment due to its excellent mechanical properties, biocompatibility and corrosion resistance.³⁷ Poor integration between bone and implant surface often leads to serious complications.³⁸ Therefore, how to promote osseointegration of bone and implant formation as early as possible is worth thinking about.

Pore structure is not only an important parameter affecting the mechanical properties of bone scaffolds, but also promotes the growth of new bone tissue into the pores, so that the implant and bone tissue form a hinged structure, which is conducive to the long-term stable fixation of the implant.³⁹ Comparative analysis of studies has shown that,^10,40_ENREF_40 large pore size promotes blood vessel growth and cell adhesion, but reduces mechanical properties; on the contrary, small pore size can enhance mechanical properties, but it is easy to cause pore clogging. In our previous work,⁹ we found that the 600 μm pore size has obvious advantages for the range and depth of the new bone tissue into the pores and the bonding strength with the implant, which is more conducive to osseointegration. Traditional techniques for fabricating porous structures (e.g., plasma spraying powders to dense substrates, using porogens and sintering titanium fibers, etc.) do not provide stability and control over the external shape and pore network of the scaffolds.^41,42 In order to solve this technical problem, 3D printing technology came into being.⁴² 3D printing can precisely control its internal structure and external shape, which helps to control the parameters of the porous structure and reduce experimental errors.⁴³ In this study, theoretical and measured values of structural parameters of porous titanium scaffolds, including porosity, pore size, cylinder size and volume. The difference between theoretical and actual structural parameters is small, indicating that the 3D printed porous titanium alloy scaffolds are of high quality.

Mechanical experiments were performed on these structures to measure their elastic modulus and yield strength values before the scaffolds were implanted in vivo. Relevant studies have shown that the elastic modulus of trabecular bone is in the range of 0.1–4.5 GPa, and the yield strength of proximal tibia and proximal femur is in the range of 0.56–55.3 MPa.⁴⁴ Studies have shown that the elastic modulus of the porous titanium alloy scaffold matches that of the host bone tissue, which can well avoid the stress shielding effect.⁴⁵ we actually measured the yield strength of the 3D printed porous titanium alloy scaffold to be about 130.5 ± 15.5Mpa and the elastic modulus to be about 2.1 ± 0.1 GPa. This indicates that the porous titanium alloy scaffolds in this study have sufficient yield strength to meet the strength requirements of orthopaedic implants and effectively reduce the risk of deformation of the implants found under physiological loads.

Unfortunately, porous titanium alloys are as biologically inert as other metallic materials.⁴⁶ Undesirable cell adhesion on the implant surface may lead to implant loosening.⁴⁷ Therefore, modifying the surface of porous titanium alloy implants to have the necessary biological activity is expected to improve the success rate of implantation.⁴⁸ As shown in Figure 2, this study simulates DOPA as a four-claw structure. The four catechol groups at one end of the DOPA peptide can undergo a strong coordination reaction with the oxide layer on the surface of the titanium alloy, and the dopatide can be firmly bound on titanium sheet. The other end is used for grafting RGD peptide and BMP-2 peptide, RGD peptide is a cell adhesion peptide, which is simulated as a green peptide chain, and BMP-2 peptide is a pro-osteogenic peptide, which is simulated as a yellow peptide chain. A chemical reaction occurs after the soaking method, which makes the two peptides bind firmly to dopatide. Bilem et al. showed that on the surface of bifunctional glass material with BMP-2 and RGD two peptides, the cell content of hBMSCs was twice that of the other two monofunctional peptide groups.⁴⁹ In this study, the titanium alloy was pickled and passivated before soaking in peptides to form a dense TiO2 film on the surface,⁵⁰ and then through a series of coordination reactions and chemical reactions, it could endow the titanium alloy with specific cell adhesion and promote osteogenesis. Bifunctionalization improves the "inertness" of the titanium alloy surface and promotes osseointegration.

As previously mentioned, the success of surface modification of the implant determines the fate of the associated osteoblasts. Therefore, after grafting different bioactive peptide coatings on porous titanium alloy scaffolds, it is necessary to use scanning electron microscopy to confirm the surface morphology of the implants.⁵¹ The results shown in Figure 5 indicate that the functionalized surface species of each group can be successfully and uniformly conjugated to the Ti6Al4V alloy. This result indicates that our covalent cross-linking method is feasible.

XPS is commonly used to study the chemical environment of various functionalized surface species.⁵² Using XPS to detect the characteristic peaks of N element on the surface of titanium alloy and quantitative analysis of N element, it is possible to judge whether the surface modification is successful or not.⁵³ It was found that the N content of group A was only 1.52%. After the surface modification of the titanium alloy scaffold, the surface element composition showed a significant enhancement of the Nls signal, indicating that the peptide was successfully grafted. Quantitative results showed that the content of N element in group B only modified by DOPA peptide was 3.89%, while the content of N element in group E modified by BMP-2 peptide and RGD peptide was the highest, which increased to 9.57%. The N element was significantly increased, which also directly proved the successful connection of DOPA peptide with the two biologically active peptides. In addition, the Ti content of group A is only 0.36%, which may be due to the thick film formed by impurities, which blocks the spectral absorption of single titanium, and does not affect the formation of TiO2 film on its surface, thus affecting the experimental results.

The manufacture of animal femoral condyle bone defect model is simple, and there are few human subjective factors. The most important thing is to directly reflect the density of new bone, the rate of new bone formation and the effect of osseointegration.⁵⁴ We implanted a titanium alloy stent into the bone tunnel of the femoral condyle of the rabbit and observed it by X-ray on the day of surgery. We found in the X-ray films that the titanium alloy scaffold was in the center of the rabbit femoral condyle, and there was no gap between the scaffold and the osseointegration. Evaluation of the effect of bone formation and integration around titanium alloy stents. The implants at the surgical site were again X-rayed at 4 and 8 weeks postoperatively. The results showed that with the prolongation of implantation time, the pores of the porous titanium alloy gradually disappeared, and the bone density around the scaffold in group E was the highest. This indicates that the bifunctional coating can effectively enhance the osteogenic and osseointegrated properties of titanium implants. In addition, when we observed the femoral specimens, we found an interesting phenomenon. The specimens of group E showed a large number of callus around the scaffold, covering the surface of the scaffold and extending beyond the bone surface, while the other groups did not have this phenomenon. It is considered that the rapid repair of the callus is due to the superior ability of the E group to promote bone growth. The positive results of this study suggest that the bifunctionalization strategy of highly biomimetic peptides will provide a facile, safe and effective means for the clinical application of titanium alloy implants.

Micro-computed tomography (Micro-CT) can accurately and quantitatively analyze factors such as the amount of bone formation, bone volume, and trabecular bone density in the sample, and can also construct three-dimensional images to intuitively reflect the effect of bone formation and osseointegration. In this part of the experiment, we used these effects of Micro-CT to scan the titanium alloy stent implanted in the body. The results showed that the BV/TV, Tb.N, Tb.Th of group E were significantly higher than the other four groups, on the contrary, Tb. Sp was significantly lower than the other four groups. This indicates that the bifunctional biologically active polypeptide coating in group E has the ability to promote the repair of bone defects, and can better promote the formation and regeneration of surrounding bone tissue. This also verifies the results of the X-ray observations.

Hard tissue sectioning technology plays a unique role in bone tissue engineering and evaluation of osseointegration status of intraosseous implants. It was found that the amount of new bone and the density of trabecular bone around the five groups of titanium alloy stents continued to increase with the prolongation of implantation time. However, the amount of new bone and the density of trabecular bone around the titanium alloy stents in groups B, C, D, and E with different proportions of bioactive peptide coatings were significantly more than those in the group A titanium alloy stent group. In particular, the dual-functional coated titanium alloy scaffold in group E adhered closely to the bone tissue, with almost no voids and fibrous tissue, while there were still gaps and more gaps between the titanium alloy scaffold and bone tissue in group A. fibrous tissue. This shows the same results as Micro-CT. It shows that the three bioactive peptide coatings can promote new bone regeneration and accelerate osseointegration, and the effect of the bifunctional coating in group E is the most significant.

In conclusion, by covalently cross-linking RGD peptide and BMP-2 peptide to the porous Ti6Al4V alloy surface, the adhesion and spreading of osteoblasts can be enhanced, further promoting osseointegration within the bone/prosthesis interface. Of course, the results of this study also have certain limitations. For example, the number of samples for statistical measurement may not be sufficient, and further in vivo experiments are required to verify whether the material is toxic to cells and to explain the mechanism of action of the RGD peptide and BMP-2 peptide as much as possible.

Conclusion

This work demonstrates that three bioactive mussel peptides can be incorporated into porous titanium alloys that effectively enhance bone growth around implants. Furthermore, bifunctional peptide coatings on porous titanium alloys can greatly enhance interfacial osteogenesis and promote osseointegration. This result confirmed the synergistic effect of the RGD peptide and the BMP-2 peptide. Therefore, the highly biomimetic peptide and bifunctionalization strategies in this work will provide a facile and effective means to improve the clinical efficacy of porous titanium alloy implants.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study is supported by Changzhou Science and Technology Project Fund. Project name: Sr/nano-ZnO/HA composite plasma spraying modified 3D printed porous Ti6Al4V antibacterial and osteopromoting activity research; project number: CQ20214029.

ORCID iD

Run Ze Zhang

References

Sidambe

. Biocompatibility of advanced manufactured titanium implants-A review. Materials 2014; 7: 8168–8188. DOI: 10.3390/ma7128168.

Kawashita

. Current progress in inorganic artificial biomaterials. J Artificial Organs: The Official Journal Jpn Soc Artif Organs 2011; 14: 163–170. DOI: 10.1007/s10047-011-0585-5.

Clavell

de Llano

Carda

, et al. In vitro assessment of the biological response of Ti6Al4V implants coated with hydroxyapatite microdomains. J Biomedical Materials Research A 2016; 104: 2723–2729. DOI: 10.1002/jbm.a.35817.

Zaharin

Abdul Rani

Azam

, et al. Effect of unit cell type and pore size on porosity and mechanical behavior of additively manufactured Ti6Al4V Scaffolds. Materials 2018; 11: 2402. DOI: 10.3390/ma11122402.

Zhang

Wang

Liu

, et al. The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration. Biomaterials 2013; 34: 3184–3195. DOI: 10.1016/j.biomaterials.2013.01.008.

Spece

Basgul

Andrews

, et al. A systematic review of preclinical in vivo testing of 3D printed porous Ti6Al4V for orthopedic applications, part I: Animal models and bone ingrowth outcome measures. J Biomedical Materials Research B, Appl Biomaterials 2021; 109: 1436–1454. DOI: 10.1002/jbm.b.34803.

Weng

Wang

, et al. Potential use of porous titanium-niobium alloy in orthopedic implants: preparation and experimental study of its biocompatibility in vitro. Plos One 2013; 8: e79289. DOI: 10.1371/journal.pone.0079289.

Deng

Liu

, et al. 3D printed Ti6Al4V bone scaffolds with different pore structure effects on bone ingrowth. J Biological Engineering 2021; 15: 4. DOI: 10.1186/s13036-021-00255-8.

Wang

Weng

Liu

, et al. 3D printing titanium alloy scaffolds with different pore sizes to repair rabbit femoral defects: 600μm pore size is more conducive to osseointegration. China Tissue Eng Res 2021; 25: 4441–4446.

10.

Ran

Yang

, et al. Osteogenesis of 3D printed porous Ti6Al4V implants with different pore sizes. J Mechanical Behavior Biomedical Materials 2018; 84: 1–11. DOI: 10.1016/j.jmbbm.2018.04.010.

11.

Diamanti

Del Curto

Pedeferri

, et al. Anodic oxidation of titanium: from technical aspects to biomedical applications. J Applied Biomaterials Biomechanics: JABB 2011; 9: 55–69. DOI: 10.5301/JABB.2011.7429.

12.

Goriainov

Cook

, Bone and metal: an orthopaedic perspective on osseointegration of metals. Acta Biomaterialia 2014; 10: 4043–4057. DOI: 10.1016/j.actbio.2014.06.004.

13.

Becker

Schmied

Kadem

, et al. Single-cell adhesion of human osteoblasts on plasma-conditioned titanium implant surfaces in vitro. J Mechanical Behavior Biomedical Materials 2020; 109: 103841. DOI: 10.1016/j.jmbbm.2020.103841.

14.

Souza

JCM

Sordi

Kanazawa

, et al. Nano-scale modification of titanium implant surfaces to enhance osseointegration. Acta Biomaterialia 2019; 94: 112–131. DOI: 10.1016/j.actbio.2019.05.045.

15.

Brubaker

Messersmith

. The present and future of biologically inspired adhesive interfaces and materials. Langmuir: The ACS Journal Surfaces Colloids 2012; 28: 2200–2205. DOI: 10.1021/la300044v.

16.

Lee

Shin

Lee

, et al. Polydopamine-mediated immobilization of multiple bioactive molecules for the development of functional vascular graft materials. Biomaterials 2012; 33: 8343–8352. DOI: 10.1016/j.biomaterials.2012.08.011.

17.

Hwang

Harrington

, et al. Mussel foot protein-1 (mcfp-1) interaction with titania surfaces. J Materials Chemistry 2012; 22: 15530–15533. DOI: 10.1039/C2JM32439C.

18.

Aye

Boyd-Moss

, et al. Scaffolds Formed via the Non-Equilibrium Supramolecular Assembly of the Synergistic ECM Peptides RGD and PHSRN Demonstrate Improved Cell Attachment in 3D. Polymers 2018; 10: 690. DOI: 10.3390/polym10070690.

19.

Wang

Shen

, et al. The functions and applications of RGD in tumor therapy and tissue engineering. Int J Mol Sci 2013; 14: 13447–13462. DOI: 10.3390/ijms140713447.

20.

Alipour

Baneshi

Hosseinkhani

, et al. Recent progress in biomedical applications of RGD-based ligand: From precise cancer theranostics to biomaterial engineering: A systematic review. J Biomedical Materials Research Part A 2020; 108: 839–850. DOI: 10.1002/jbm.a.36862.

21.

Yang

Zhang

Liu

, et al. Function and Mechanism of RGD in Bone and Cartilage Tissue Engineering. Front Bioengineering Biotechnology 2021; 9: 773636. DOI: 10.3389/fbioe.2021.773636.

22.

Bellis

. Advantages of RGD peptides for directing cell association with biomaterials. Biomaterials 2011; 32: 4205–4210. DOI: 10.1016/j.biomaterials.2011.02.029.

23.

Halloran

Durbano

Nohe

, et al. Bone Morphogenetic Protein-2 in Development and Bone Homeostasis. J Developmental Biology 2020; 8: 19. DOI: 10.3390/jdb8030019.

24.

Bragdon

Moseychuk

Saldanha

, et al. Bone morphogenetic proteins: a critical review. Cell Signalling 2011; 23: 609–620. DOI: 10.1016/j.cellsig.2010.10.003.

25.

Zhou

Xia

, et al. Loading BMP-2 on nanostructured hydroxyapatite microspheres for rapid bone regeneration. Int Journal Nanomedicine 2018; 13: 4083–4092. DOI: 10.2147/IJN.S158280.

26.

James

LaChaud

Shen

, et al. A Review of the Clinical Side Effects of Bone Morphogenetic Protein-2. Tissue engineering Part B. Reviews 2016; 22: 284–297. DOI: 10.1089/ten.TEB.2015.0357.

27.

Agarwal

Garcia

. Biomaterial strategies for engineering implants for enhanced osseointegration and bone repair. Adv Drug Delivery Reviews 2015; 94: 53–62. DOI: 10.1016/j.addr.2015.03.013.

28.

Chien

Tsai

Liu

, et al. Noggin Inhibits IL-1beta and BMP-2 Expression, and Attenuates Cartilage Degeneration and Subchondral Bone Destruction in Experimental Osteoarthritis. Cells 2020; 9: 927. DOI: 10.3390/cells9040927.

29.

Zhao

Huang

Zhang

, et al. Mussel-Inspired Peptide Coatings on Titanium Implant to Improve Osseointegration in Osteoporotic Condition. ACS Biomaterials Science Engineering 2018; 4: 2505–2515. DOI: 10.1021/acsbiomaterials.8b00261.

30.

Pan

Sun

Zhang

, et al. Biomimetic Design of Mussel-Derived Bioactive Peptides for Dual-Functionalization of Titanium-Based Biomaterials. J Am Chem Soc 2016; 138: 15078–15086. DOI: 10.1021/jacs.6b09770.

31.

Gao

Liu

, et al. Effects of immediately static loading on osteointegration and osteogenesis around 3D-printed porous implant: A histological and biomechanical study. Mater Science Engineering C, Mater Biological Applications 2020; 108: 110406. DOI: 10.1016/j.msec.2019.110406.

32.

Mehta

Schmidt-Bleek

Duda

, et al. Biomaterial delivery of morphogens to mimic the natural healing cascade in bone. Adv Drug Delivery Reviews 2012; 64: 1257–1276. DOI: 10.1016/j.addr.2012.05.006.

33.

Harris

Bemenderfer

Wessel

, et al. A review of mouse critical size defect models in weight bearing bones. Bone 2013; 55: 241–247. DOI: 10.1016/j.bone.2013.02.002.

34.

Bakhtiary

Liu

Ghorbani

, et al. Bioactive Inks Development for Osteochondral Tissue Engineering: A Mini-Review. Gels 2021; 7: 274. DOI: 10.3390/gels7040274.

35.

Gruskin

Doll

Futrell

, et al. Demineralized bone matrix in bone repair: history and use. Adv Drug Delivery Reviews 2012; 64: 1063–1077. DOI: 10.1016/j.addr.2012.06.008.

36.

Xiao

Chen

Biao

, et al. Bio-functionalization of biomedical metals. Mater Science Engineering C, Mater Biological Applications 2017; 70: 1057–1070. DOI: 10.1016/j.msec.2016.06.067.

37.

Yang

Zhao

, et al. New Developments of Ti-Based Alloys for Biomedical Applications. Materials 2014; 7: 1709–1800. DOI: 10.3390/ma7031709.

38.

Carragee

Hurwitz

Weiner

, et al. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine Journal : Official Journal North Am Spine Soc 2011; 11: 471–491. DOI: 10.1016/j.spinee.2011.04.023.

39.

Yuan

Chandrakar

, et al. 3D porous Ti6Al4V-beta-tricalcium phosphate scaffolds directly fabricated by additive manufacturing. Acta Biomaterialia 2021; 126: 496–510. DOI: 10.1016/j.actbio.2021.03.021.

40.

Reznikov

Chase

Ben Zvi

, et al. Inter-trabecular angle: A parameter of trabecular bone architecture in the human proximal femur that reveals underlying topological motifs. Acta Biomaterialia 2016; 44: 65–72. DOI: 10.1016/j.actbio.2016.08.040.

41.

Zhao

Wang

, et al. Microfluidic Technology for the Production of Well-Ordered Porous Polymer Scaffolds. Polymers 2020; 12: 1863. DOI: 10.3390/polym12091863.

42.

Trevisan

Calignano

Aversa

, et al. Additive manufacturing of titanium alloys in the biomedical field: processes, properties and applications. J Applied Biomaterials Functional Materials 2018; 16: 57–67. DOI: 10.5301/jabfm.5000371.

43.

Wang

Liu

, et al. Metal material, properties and design methods of porous biomedical scaffolds for additive manufacturing: A review. Front Bioengineering Biotechnology 2021; 9. DOI: 10.3389/fbioe.2021.641130.

44.

Heary

Parvathreddy

Sampath

, et al. Elastic modulus in the selection of interbody implants. J Spine Surgery 2017; 3: 163–167. DOI: 10.21037/jss.2017.05.01.

45.

Peng

Liu

Jiang

, et al. Bionic mechanical design and 3D printing of novel porous Ti6Al4V implants for biomedical applications. J Zhejiang Univ Sci B 2019; 20: 647–659. DOI: 10.1631/jzus.B1800622.

46.

Gao

Fan

, et al. Biofunctional magnesium coated Ti6Al4V scaffold enhances osteogenesis and angiogenesis in vitro and in vivo for orthopedic application. Bioactive Materials 2020; 5: 680–693. DOI: 10.1016/j.bioactmat.2020.04.019.

47.

Mjoberg

. Hip prosthetic loosening: A very personal review. World Journal Orthopedics 2021; 12: 629–639. DOI: 10.5312/wjo.v12.i9.629.

48.

Bijukumar

McGeehan

Mathew

, et al. Regenerative medicine strategies in biomedical implants. Curr Osteoporosis Reports 2018; 16: 236–245. DOI: 10.1007/s11914-018-0441-0.

49.

Bilem

Chevallier

Plawinski

, et al. RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells. Acta Biomaterialia 2016; 36: 132–142. DOI: 10.1016/j.actbio.2016.03.032.

50.

Mohan

Durgalakshmi

Geetha

. Electrophoretic deposition of nanocomposite (HAp+ TiO2) on titanium alloy for biomedical applications. Ceramics Int 2012; 38: 3435–3443.

51.

Eghiaian

Rico

Colom

, et al. High-speed atomic force microscopy: imaging and force spectroscopy. FEBS Letters 2014; 588: 3631–3638. DOI: 10.1016/j.febslet.2014.06.028.

52.

Zhao

Gao

Pujari

, et al. Universal calibration of computationally predicted N 1s binding energies for interpretation of XPS experimental measurements. Langmuir: The ACS Journal Surfaces Colloids 2017; 33: 10792–10799. DOI: 10.1021/acs.langmuir.7b02301.

53.

Cools

Asadian

Nicolaus

, et al. Surface Treatment of PEOT/PBT (55/45) with a Dielectric Barrier Discharge in Air, Helium, Argon and Nitrogen at Medium Pressure. Materials 2018; 11: 391. DOI: 10.3390/ma11030391.

54.

Guo

Kim

Orchard

, et al. A Rabbit Femoral Condyle Defect Model for Assessment of Osteochondral Tissue Regeneration. Tissue Engineering C Methods 2020; 26: 554–564. DOI: 10.1089/ten.TEC.2020.0261.