Bone Marrow Mesenchymal Stem Cells (BMSc) primary cells (HUM-iCell-s011, iCell Bioscience, Shanghai, China) were passed the mycoplasma and Short Tandem Repeat (STR) certification, then cultured, passaged and frozen in liquid nitrogen for spare parts to obtain the appropriate titer of Flag-tagged BMP-9 recombinant adenovirus for transfection of BMSc cells, and Flag-tagged BMP-9 recombinant adenovirus (Ad-BMP-9) was used to transfect BMSc cells.

Cells in good growth status, after reaching 80% density, were washed with Phosphate Buffered Saline (PBS) (10010002, Gibco™, Waltham, MA, USA) in culture flasks, added with 0.25% trypsin digest, incubated at 37 °C for 2 minutes, gently shaken to dislodge all cells, centrifuged by adding 3 mL of medium, discarded the supernatant, and blown up into cell suspension. Subsequently, the cells were counted on a hemocyte counting plate, and the cell density was adjusted to 2 $\times$ 10⁵ cells/mL for spreading.

The cells were grouped after attaching to the wall. (1) Ad-vector: the cell fusion rate reached about 50%, change fresh medium 1h in advance, use empty adenovirus, infect the cells with 20 µL of adenovirus in T25 cell bottles for 48 hours, and observe the cells under the microscope after the change of the solution; (2) Overexpression group (Ad-BMP9): the cell fusion rate reached about 50%, change fresh medium 1h in advance, use Flag-tagged BMP-9 adenovirus to infect one T25 cell bottle with 20 µL for 48 hours, and observe the cells under the microscope (BLD-200, COSSIM, Beijing, China).

BMSc cells that had been transfected with Ad-BMP-9 were taken to spread 80% of the bottom of the dish and centrifuged after trypsin termination. Remove the supernatant, add complete medium to resuspend, and adjust the cell concentration to 2 $\times$ 10⁶ cells/mL. 3 mL of liquid GT (G0040, Solarbio, China) was taken in a 6-well plate, and aspirate 2 mL of the above Ad-BMP-9/BMSc cell suspension slowly and dropwise added to liquid GT to make Ad-BMP-9/BMSc/GT, and set aside.

MG63 cells (CRL-1427, ATCC, Manassas, VA, USA) were authenticated by STR profiling and confirmed to be free of mycoplasma contamination. Cells were harvested by trypsin digestion, counted, and adjusted to a density of 2 $\times$ 10⁵ cells/mL. Ad-BMP-9/BMSc/GT was then co-cultured with MG63 cells for 10 days.

Group culture: (1) Ad-vector/BMSc/GT+MG63 cell co-culture control group; (2) Ad-BMP-9/BMSc/GT+MG63 cell co-culture group. GT was used as the control, and the proliferation and differentiation of Ad-BMP-9/BMSc/GT-promoted osteoblasts were observed by inverted biomicroscope (CKX3-SLP, OLYMPUS, Japan) morphology observation, Cell Counting Kit-8 (CCK-8), and human alkaline phosphatase (ALP) activity assay on the 1st, 5th, and 10th days, respectively.

The n-CDHA/PAA composite biomaterial discs with 30% n-CDHA mass ratio were prepared by in situ polymerization method, and the size of the discs was $\phi$6 $\times$ 2 mm. n-CDHA/PAA composite biomaterials with multilayers of Ad-BMP-9/BMSc/GT coating were prepared as follows. Step 1: Ad-BMP-9/BMSc cell suspension with a cell concentration of 2 $\times$ 10⁶ cells/mL was taken and slowly added dropwise to the surface of n-CDHA/PAA discs after freeze-drying using liquid nitrogen (first layer coating); Step 2: Liquid GT with a thickness of about 1 mm was coated on the first layer coating after freeze-drying using liquid nitrogen (second layer coating); Step 3: The first and second steps above were repeated to make the multilayer Ad-BMP-9/BMSc/GT coated n-CDHA/PAA composite biomaterial.

According to the experimental design the following three experimental subgroups were designed: (1) n-CDHA/PAA control group (without any coating treatment); (2) control group of composite biomaterials of n-CDHA/PAA with single-layer coated GT; and (3) experimental group of composite biomaterials of multilayered Ad-BMP-9/BMSc/GT coated n-CDHA/PAA. After co-culturing the materials of the experimental and control groups with well-grown MG63 cells for 10 days, the cell culture medium was aspirated at day 1, 5, and 10, respectively, followed by two washes with PBS. According to the protocols of the Cell Plasma Membrane Staining Kit with DiO (Green Fluorescence) (C1993S, Beyotime, Shanghai, China), an appropriate volume of green fluorescent cell membrane staining working solution was added and gently agitated to ensure uniform staining of all cells. The cells were incubated at 37 °C in the dark for 20 minutes. The cell membrane staining working solution was then aspirated, and the cells were washed three times with PBS. Finally, 37 °C pre-warmed cell culture medium was added, and the adhesion and growth of MG63 cells on the material surface were observed under an inverted biological microscope. The proliferation of MG63 cells and ALP activity were observed by CCK-8 and ALP activity analysis at three time points, respectively; and the proliferation of MG63 cells and the activity of ALP were detected by Quantitative Polymerase Chain Reaction (q-PCR) and Western Blot (WB), respectively, in the MG63 cells.

Cell proliferation of MG63 cells was assessed using a CCK-8 assay (C0038, Beyotime, China) at three time points (days 1, 5, and 10). Cells were seeded in 96-well plates, and 10 µL of CCK-8 solution was added into each well. Blank wells containing only cell culture medium and CCK-8 solution were used as zero controls. Plates were incubated in a cell culture incubator (BPH-9042, Yiheng, Shanghai) for 1 hour. Absorbance was measured at 450 nm using an enzyme marker (CMax Plus, Molecular Devices, San Jose, CA, USA).

Following the procedure described in the manual, the ALP enzyme-linked immunosorbent assay (ELISA) kit (CB12388-Hu, COIBO, Shanghai, China) was used to perform cell activity analysis of MG63 cells at three different time points (Day 1, Day 5, and Day 10). The cell suspension was diluted with phosphate-buffered saline (PBS). Cells were lysed and intracellular components were released through repeated freezing and thawing. After centrifugation for 20 minutes, the supernatant was collected. Standard wells, sample wells, and blank wells were set up accordingly. A standard curve was generated using different concentrations of the standard in the standard wells. Sample wells were loaded with 10 µL of cell lysate, followed by the addition of 100 µL of the enzyme-labeled reagent. After incubation at 37 °C for 60 minutes, the wells were washed five times with diluted washing solution. The chromogenic agent was added, and color development was allowed proceed in the dark at 37 °C for 15 minutes. Following reaction termination, the absorbance (OD value) was measured at 450 nm using an enzyme-linked immunosorbent assay reader (CMax Plus, Molecular Devices, USA).

Cell lysates were prepared from each group using Radioimmunoprecipitation Assay (RIPA) buffer (P0013B, Beyotime, China) and centrifuged to collect the supernatant. Total protein was extracted and stored at –80 °C for subsequent Western blotting. For Western blotting, 500 µg of total protein from each sample was mixed with 5$\times$SDS sample buffer at a 4:1 ratio and denatured by heating at 100 °C for 6 minutes. Sixty micrograms of denatured protein were loaded onto a 10% SDS-PAGE gel (PG212, Epizyme, Shanghai, China) and transferred to a polyvinylidene fluoride membrane (PVDF) membrane (10600023, Amersham, Germany). The membrane was blocked with a 5% skim milk solution for 1 hour, washed three times with Tris-Buffered Saline with Tween 20 (TBST) buffer (5 minutes each wash), and incubated overnight at 4 °C with primary antibodies against DDDDK-Tag (AE169, abclonal, China) and BMP-9 (abclonal, A10537, China) and GAPDH (abclonal, A19056, China) at a 1:1000 dilution. The DDDDK-Tag is an antibody specifically targeting the Flag tag. After three cycles of washing, the membrane was incubated with a secondary antibody (abclonal, AS014, China) at room temperature for 1 hour, followed by three additional washes with TBST. Signal detection was performed using an ECL enhanced chemiluminescence detection kit (34580, Thermo, Waltham, MA, USA). GAPDH expression was regarded as an endogenous control for normalization. Each sample’s Western blotting experiment was repeated at least three times.

Total RNA was extracted from BMSc and MG63 cells using TsingZol (TsingKe, Beijing, China) and quantified using agarose gel electrophoresis and Nanodrop oneC (Thermo, USA). Only RNA samples meeting quality criteria were used for subsequent analysis. First-strand cDNA was synthesized using the GoldenstarTM RT6 cDNA Synthesis Kit Ver.2 (TsingKe, Beijing, China). The qPCR experiments were conducted using the 2$\times$T5 Fast qPCR Mix (SYBR Green I) (TsingKe, Beijing, China), with GAPDH mRNA serving as the endogenous reference gene for normalization. All procedures were performed according to the manufacturer’s protocols. Relative gene expression levels were analyzed using the 2^{-Δ⁢Δ⁢Ct} method, with each result repeated at least three times. Specific primers are listed in Table 1.

Twenty-four male New Zealand White rabbits, weighing between 2 kg to 2.2 kg, were provided by Byrness Weil biotech Ltd (Chongqin, China). The animal room maintained a 12-hour light/dark cycle, allowing free access to water and food for the experimental rabbits, with a temperature maintained between 23–25 °C. All experimental procedures and animal care were strictly conducted in accordance with the “Guide for the Care and Use of Laboratory Animals”, and were approved by the Ethical Committee of Chongqing Medical Hospital (IACUC-CQMU-2023-0067). After one week of adaptive feeding, the animals entered the experiment.

A femoral condyle fracture model was constructed in 24 New Zealand White rabbits. Anesthesia was induced by administering 3% pentobarbital sodium (1 mL/kg) via ear vein injection. The animals were positioned supine and fixed. All surgical procedures were conducted under strict aseptic conditions. A 5 cm longitudinal parapatellar incision was made, and the subcutaneous tissue, fascia, and muscles were dissected to expose the patella. After dislocation of the patella, the femoral condyle was exposed. An intercondylar femoral fracture was created using a saw. Following temporary reduction, a 2.5 mm diameter hole was drilled from the lateral side to the opposite cortex. Two different types of screws were used to fix the left and right legs of each rabbit. Specifically, the experimental group received multilayer Ad-BMP-9/BMSc/GT coated n-CDHA/PAA composite biomaterials screws. Two control groups were treated with medical metal screws and n-CDHA/PAA composite biomaterials screws, respectively.

Rabbits were sacrificed at 1, 4, 12, and 24 weeks post-surgery via intravenous air injection. Operated knee joints were harvested and stored in liquid nitrogen for subsequent analysis. Digital veterinary X-ray imaging systems (20111337C, Hangzhou Zhiyuan Medical Equipment Co., Ltd., Hangzhou, China) were used to take X-ray images of the surgical sites of the living rabbits at different time points (kVP: 58, mAs: 4.00, mA: 320). Micro-CT in vivo imaging system for small animals (IMAGING 100, Hefei Ruishi Medical Technology Co., Ltd., Hefei, China) was used to observe the material-bone interface integration at four time points and to measure the bone density adjacent to the interface. Scanning electron microscopy (SU8100, Hitachi, Japan) was used to observe the bone-screw interface of the rabbit knee joints in each group (3.0 kV $\times$ 1.0 k), and an energy-dispersive X-ray spectrometer attached to the scanning electron microscope was used to analyze the calcium deposition at the material-bone interface. Biomechanical testing was conducted to measure the interface binding strength between the material and bone tissue at four time points. The surface condition of the screws after push-out experiments at four time points was observed using scanning electron microscopy (SU8100, Hitachi, Tokyo, Japan).

The sample was fixed with 4% paraformaldehyde, decalcified with Ethylenediaminetetraacetic acid (EDTA) (DD0002, Leagene, Beijing, China), dehydrated with ethanol (64-17-5, Chuandong Chemical, Chongqing, China), transparent with xylene (1330-20-7, Chuandong Chemical, Chongqing, China), waxed in paraffin (80200-0007, Shitai, Hangzhou, China), then embedded, sliced and baked. The slices were dewaxed in xylene, dehydrated in ethanol, dipped in toluidine blue dye solution (G1032, Servicebio, Wuhan, China) for 5 min, washed, and differentiated into light blue background with 0.1% glacial acetic acid, and stopped by tap water washing. After dehydration and sealing, the slices were observed and recorded with Mshot MF53 microscope (Mingmei, Guangdong, China).

The slices were processed by Masson trichromatic staining kit (Solarbio, Beijing, China) according to the instructions. The steps are as follows: Dip the prepared paraffin slices into mordant dye solution, let them act at room temperature for one night, and rinse them with running water. The sections were stained with lapis lazuli blue staining solution for 3 min, Mayer hematoxylin staining solution for 3 min, acidic ethanol differentiation solution until the tissues turned red completely, and washed with distilled water. The slices were dyed with ponceau magenta dye solution for 10 min, treated with phosphomolybdic acid solution for 10 min, and dyed with aniline blue dye solution for 5 min. After washing the slices with weak acid solution to remove aniline blue solution, continue to drip weak acid working solution to cover the slices for 2 min. The samples were dehydrated and sealed, then observed and recorded under a microscope (MF53, Mingmei, Guangzhou, China).

The sections were processed using the Eosin-Van Gieson (EVG) staining kit (Servicebio, Wuhan, China) according to the instructions, as follows: The paraffin sections were dyed with 5:2:2 mixed EVG A, B and C solutions for 5 min, and then dyed with EVG B dye solution diluted twice to control the differentiation until the elastic fibers were dark purple and the background was grayish white under the microscope. The EVG dyes D and E were mixed at a ratio of 1:9, and the slices were dyed for 12 min and washed with water. Finally, the slices were dehydrated and sealed, and observed and recorded under a microscope (MF53, Mingmei, China).

Experimental data were presented as mean $\pm$ standard error, and comparisons between groups were performed using the T-test, and GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA) was used for data analysis and visualization. Differences were considered statistically significant at p 0.05.

Firstly, we successfully established BMP-9 overexpressing BMSc cells by transfecting the Flag-tagged BMP-9 recombinant adenovirus. Successful transfection of BMSc cells was confirmed by a significant increase in both BMP-9 gene and DDDDK-Tag protein expression levels, as depicted in Fig. 1A,B (p 0.05). Subsequently, we constructed Ad-BMP-9/BMSc/GT by loading Ad-BMP-9/BMSc onto GT. Ad-BMP-9/BMSc/GT was co-cultured with MG63 cells for 10 days, and the cell proliferation and differentiation capabilities were assessed on days 1, 5, and 10, respectively. Ad-BMP-9/BMSc/GT significantly enhanced both MG63 cell proliferation and ALP activity compared to the Ad-vector/BMSc/GT group (Fig. 1C,D, p 0.05), suggesting that Ad-BMP-9/BMSc/GT promotes both proliferation and differentiation of MG63 cells. Furthermore, we monitored the changes in BMP-9 gene and protein expression levels in MG63 cells. The results showed that the BMP-9 mRNA and protein expression levels in MG63 cells of the Ad-BMP-9/BMSc/GT group gradually increased over time (Fig. 1E,F, p 0.05), which is consistent with the promotion of MG63 cell proliferation and differentiation.

Fig. 1.

The Effect of Gelatin (GT)-Loaded Adenovirus (Ad)-Bone Morphogenetic Protein 9(BMP-9)/Bone Marrow Mesenchymal Stem Cells (BMSc) on the Proliferation and Differentiation of MG63 Cells. (A) Expression of the BMP-9 gene and (B) DDDDK-Tag protein after transfection of BMSc cells with recombinant adenovirus (Ad-BMP-9) for 48 hours. (C) Cell viability assays performed on days 1, 5, and 10 during the 10-day co-culture of Ad-BMP-9/BMSc/GT with MG63 cells. (D) alkaline phosphatase (ALP) activity assay. (E) Detection of BMP-9 gene levels in cells on days 1, 5, and 10. (F) Relative expression of BMP-9 protein in cells on days 1, 5, and 10. *, Indicates a statistically significant difference between the two groups with p-value 0.05; **, p-value 0.01; ***, p-value 0.001; ****, p-value 0.0001. n = 3.

To evaluate the in vitro biocompatibility of the multilayer Ad-BMP-9/BMSc/GT coated n-CDHA/PAA composite biomaterials, we conducted cell cultures on the experimental group with the multilayer Ad-BMP-9/BMSc/GT coating on n-CDHA/PAA composite biomaterials (Ad-BMP-9/GT coating), the control group with a single layer of GT coating on n-CDHA/PAA composite biomaterials (GT coating), and the n-CDHA/PAA control group (Substrate), with a series of assessments performed on days 1, 5, and 10. To visually observe the growth of cells on the material surface, we conducted microscopic observations. The results showed that on the first day, there was not much cell growth in all groups. In the Ad-BMP-9/GT coating group, MG63 cells exhibited good spreading and growth morphology on the material surface, with a significant increase in cell numbers (Fig. 2A). Concurrently, CCK-8 assay results indicated that the proliferation capacity of MG63 cells in the Ad-BMP-9/GT coating group was significantly enhanced compared to the Substrate group, and this capacity continued to improve over time (Fig. 2B, p 0.05). Furthermore, ALP activity assay results showed that the ALP activity of MG63 cells in the Ad-BMP-9/GT coating group was markedly higher than that of the control group, indicating that the Ad-BMP-9/GT coating can promote the differentiation of MG63 cells (Fig. 2C, p 0.05). Lastly, we detected the mRNA and protein expression levels of BMP-9 in MG63 cells. The results showed that compared to the Substrate group, the mRNA and protein expression levels of BMP-9 in MG63 cells in the Ad-BMP-9/GT coating group were significantly elevated (Fig. 2D,E, p 0.05), and these expression levels gradually increased over time. This suggests that the Ad-BMP-9/GT coating can promote the proliferation and differentiation of MG63 cells by continuously releasing BMP-9.

Fig. 2.

Assessment of Biocompatibility of Multilayer Ad-BMP-9/BMSc/GT Coated nano-calcium deficient hydroxyapatite/poly-amino acid(n-CDHA/PAA) Composite Biomaterials with MG63 Cells. (A) Microscopic observation of MG63 cell growth on the material surface at three time points. Scale bars: 100 µm. (B) Cell Counting Kit-8 (CCK-8) assay for the proliferation of MG63 cells at three time points. (C) ALP activity assay for the viability of MG63 cells at three time points. (D) Quantitative Polymerase Chain Reaction (q-PCR) detection of BMP-9 mRNA expression in MG63 cells at three time points. (E) Western blot (WB) detection of BMP-9 protein expression in MG63 cells at three time points. ***, Indicates a statistically significant difference between groups with p-value 0.001. n = 3.

In vitro experimental results indicate that the multilayer Ad-BMP-9/BMSc/GT coated n-CDHA/PAA composite biomaterials exhibit good biocompatibility with MG63 cells and effectively promote the attachment, proliferation, differentiation, and expression of BMP-9 in these cells. Subsequently, we constructed an intercondylar femoral fracture model in rabbits and fixed it with Ad-BMP9/GT coated n-CDHA/PAA composite screws, medical metal screws, and n-CDHA/PAA composite screws, followed by a series of assessments at 1, 4, 12, and 24 weeks postoperatively. Micro-CT images revealed that at one week post-surgery, there were clear fracture gaps visible in the knee joint samples of all groups; at four weeks post-surgery, the fracture gaps were less distinct compared to the first week, indicating that new bone formation occurs over time; at twelve weeks post-surgery, the fracture gaps had essentially healed, with no obvious signs of fracture visible in the Micro-CT images, and no abnormalities in the overall samples; the bone density of the knee joint samples in all groups gradually increased over time, with the Ad-BMP9/GT coated n-CDHA/PAA composite screws showing the best results at later stages (Fig. 3A,B, p 0.05). Biomechanical testing results showed that medical metal screws had certain advantages in terms of maximum load during early bone fixation; as the duration of screw fixation extended, the fixation strength of the composite biomaterials screws coated with the multilayer Ad-BMP-9/BMSc/GT coating was higher than other two groups, indicating that Ad-BMP9/GT coated n-CDHA/PAA composite screws have good osseointegration capabilities, which help to enhance the integration of neo-tissue at the bone wound and the screw surface (Fig. 3C, p 0.05). SEM observations showed that at one week post-surgery, there was a small amount of neo-tissue at the bone interface in all groups; at four weeks post-surgery, the amount of neo-tissue at the bone interface increased in all groups, but cracks were observed; at twelve weeks post-surgery, there was more neo-tissue at the bone interface, which was more tightly packed, especially in the Ad-BMP9/GT coated n-CDHA/PAA composite screws group where a clear three-dimensional reticular structure was visible, indicating that the multilayer Ad-BMP-9/BMSc/GT coating on the composite biomaterials screws aids in the recovery after bone trauma; the SEM results at twenty-four weeks post-surgery showed that the bone interfaces in all groups had recovered well, indicating that the materials implanted long-term in animals had no side effects (Fig. 4).

Fig. 3.

Assessment of the Integration of Multilayer Ad-BMP-9/BMSc/GT Coated n-CDHA/PAA Composite Biomaterials with Neo-tissue at Rabbit Bone Wounds. (A) Micro-CT imaging to observe the integration at the composite biomaterial-bone interface at four time points (1 week, 4 weeks, 12 weeks, 24 weeks), red represents cortical bone, and green represents trabecular bone. (B) Measurement of bone density adjacent to the composite biomaterial-bone interface. (C) Biomechanical testing to detect the interface binding strength between the composite biomaterials and bone tissue at 1 week, 4 weeks, 12 weeks, and 24 weeks. *, Indicates a statistically significant difference between groups with p-value 0.05; **, p-value 0.01; ***, p-value 0.001. n = 3.

Fig. 4.

Scanning electron microscopy (SEM) to observe the interface between the composite biomaterials and bone tissue at 1 week, 4 weeks, 12 weeks, and 24 weeks. Red arrow: three-dimensional structure; Blue arrow: crack. Scale bars: 50 µm.

X-ray images of the surgical sites of the rabbit femur show that none of the screws in any group exhibited loosening or detachment (Fig. 5A). Starting from the 4th week, obvious neo-tissue attachment was observed on the surface of the screws in all groups. By the 12th week, the neo-tissue was more tightly integrated with the screw surfaces, with the Ad-BMP9/GT coated n-CDHA/PAA composite screws showing the most abundant new bone tissue, visible cells closely adhering to the material surface. By the 24th week, the surfaces of the screws in all groups were covered with biological tissue, indicating that over time, there was no rejection phenomenon between the biological body and the materials (Fig. 5B). Calcium deposition was significantly enhanced at the material-bone interface in Ad-BMP9/GT coated n-CDHA/PAA composite screws compared to medical metal screws at both 12 w and 24 w (Fig. 5C, p 0.05). Further supporting the ability of multilayer Ad-BMP-9/BMSc/GT coated n-CDHA/PAA composite biomaterials to promote bone tissue regeneration.

Fig. 5.

The Impact of Multilayer Ad-BMP-9/BMSc/GT Coated n-CDHA/PAA Composite Biomaterials on Neo-tissue Formation at Rabbit Bone Wounds. (A) X-ray imaging (anteroposterior view) of screw loosening at the surgical sites of each group at 1 week, 4 weeks, 12 weeks, and 24 weeks post-surgery. (B) Scanning electron microscopy (SEM) observation of the screw surface conditions after push-out experiments at 1 week, 4 weeks, 12 weeks, and 24 weeks. Scale bars: 50 µm. (C) X-ray energy dispersive spectroscopy (EDS) analysis of calcium deposition at the material-bone interface at 1 week, 4 weeks, 12 weeks, and 24 weeks. **, Indicates a statistically significant difference between groups with p-value 0.01; ***, p-value 0.001. n = 3. Red arrow: three-dimensional structure.

To determine the effect of Ad-BMP-9/BMSc/GT Coated n-CDHA/PAA Composite Biomaterials on chondroitin sulfate and collagen fiber growth in bone tissue, we observed rabbit bone wounds by multiple staining.

Based on the metachromatic property of chondroitin sulfate, which stains purple-red with toluidine blue, we conducted measurements of the staining results, including the area, depth of the stained regions, and their proportions in the total area. Statistical analysis revealed no significant differences in chondroitin sulfate content among the groups at weeks 1 and 4. However, at week 12, the chondroitin sulfate content in the bone tissue of the Ad-BMP-9/BMSc/GT coated n-CDHA/PAA group was significantly higher than that in the n-CDHA/PAA group (p 0.05). Furthermore, at both weeks 24, the chondroitin sulfate content in the Ad-BMP-9/BMSc/GT coated n-CDHA/PAA group was significantly higher than that in the Medical metal screw group (Fig. 6, p 0.05).

Fig. 6.

The Impact of Multilayer Ad-BMP-9/BMSc/GT Coated n-CDHA/PAA Composite Biomaterials on Chondroitin Sulfate at Rabbit Bone Wounds by Toluidine Blue Staining. *, Indicates a statistically significant difference between groups with p-value 0.05; **, p-value 0.01. n = 3. Scale bars: 50 µm.

The results of Massonstaining and Verhoeff’s vangiesonstaining showed that there was no significant difference in the growth of collagen fibers in the bone tissue of each group at the 1th and 4th weeks, but at the 12th and 24th weeks, Ad-BMP-9/GT Coated n-CDHA/PAA composite screws group exhibited the most abundant growth of collagen fibers in the bone tissue (Fig. 7, p 0.05).

Fig. 7.

The Impact of Multilayer Ad-BMP-9/BMSc/GT Coated n-CDHA/PAA Composite Biomaterials on Collagen Fiber Growth at Rabbit Bone Wounds. (A) Masson staining. (B) Verhoeff’s van gieson staining. *, Indicates a statistically significant difference between groups with p-value 0.05; **, p-value 0.01. n = 3. Scale bars: 50 µm.