To prepare PHBV-mPEG5k (PP5), mPEG-5k (Sigma, China) was dissolved in dry dichloromethane. The solution was then heated to 80°C under vacuum for 24 h and subsequently cooled to room temperature. The mPEG-5k solution was then added dropwise to the hexamethylene diisocyanate solution with stirring and the mixture was left to react in the dark at room temperature for 6 h to obtain mPEG5k-NCO. Next, PHBV (Tianan Biologic Material Ltd., China) was dissolved in dry dichloromethane and heated to 80°C under vacuum for 5 h and subsequently cooled to room temperature. The mPEG5k-NCO solution was then added dropwise to the PHBV solution and the mixture was left to react in the dark at room temperature for 3 days. The crude product was obtained by rotary evaporation to remove the dichloromethane. The resulting product was then purified by dissolving it in tetrahydrofuran and gradually adding distilled water with stirring. After filtration and washing with distilled water, the purified product was dried under vacuum to obtain PHBV-PEG5k (PP5).

For preparation of PP5 scaffold, PP5 was dissolved in deionized water and heated to 50°C for 24 h, until the polymer was completely dissolved, transferring the solution to 6 mm cell culture dish and stored at −20°C for 12 h. Finally, the solution was lyophilizated at −20°C, the scaffold was obtained.

The Fourier transform infrared spectra of mPEG5k, PHBV and PP5 were recorded using a Perkin Elmer spectrophotometer (spectrum 2) through the solid-state KBr pellet method within the range of 4,000–400 cm⁻¹.

The ¹H-NMR of the polymer was recorded using FT-NMR JEOL AL 500 FT-NMR Proton nuclear magnetic resonance spectrometer in CDCl₃. Tetramethylsilane was used as an internal reference.

A series of GelMA (SunP Gel G1) gels with varying concentration (0.25% w/v, 0.5% w/v, 1% w/v, 1.5% w/v and 2% w/v) were prepared to find the optimal concentration for loading MT. A mixture of 0.05 gMT and different masses of GelMa were added to 10 mL Phosphate Buffer Saline (PBS), and stirred at 650 r/min for 2 h in a 40°C water bath until fully dissolved. Then the MT solution was fully absorbed by PP5 scaffold and placed at room temperature (23–25°C) until it underwent liquid-solid conversion, resulting in the production of MT-loaded PP5 scaffold.

To prepare the MT solution, a mixture of absolute ethanol and chloroform (V: V = 9:1) was used as the solvent. The maximum absorption peak was measured using an ultraviolet-visible spectrophotometer (Mettler Toledo, Switzerland) within a wavelength range of 200–350 nm. A standard curve was drawn by fitting the absorbance with the mass concentration of the MT solution at the wavelength of the maximum absorption peak (278 nm) using linear regression. Freeze-dried MT-loaded GelMa gel (50 mg) was dissolved in PBS (10 mL) and centrifuged at 12,000 rpm for 5 min. Then, 0.5 mL of the supernatant was diluted in PBS in a 25 mL volumetric flask and the absorbance value of the solution was measured at 278 nm. The mass of melatonin was calculated from the standard curve and the LE and LC was calculated by following formula: In vitro scaffold drug release study L E = m a / m b × 100 % ; L C = m a / m c × 100 % where m_a means the mass of melatonin in MT-loaded GelMA gel, m_b represents total mass of MT and m_c is the mass of GelMA in MT-loaded GelMA gel.

Standard curve of MT was first drawn using PBS (pH = 7.4) as solvent under the wavelength of the maximum absorption peak (278 nm). MT-loaded GelMA (100 mg) was dissolved in PBS and placed in a dialysis bag. The dialysis bag was then placed in a beaker containing the same pH PBS buffer and was vibrated horizontally in a constant temperature shaker (37°C ± 0.5°C) with a vibration frequency of 50 times/min. The release liquid (5 mL) was replaced with same amounts of fresh PBS at selected time intervals. The absorbance value of the solution was measured at 278 nm and the drug release rate was calculated as follow: R % = ρ n V + V i ∑ t n − i ρ i M D × 100 where R means the cumulative drug release rate (%); n means the number of samplings. (%) represents the mass concentration (g/L) of the drug in the nth release. V represents the total volume of the release liquid. ρ i represents the mass concentration (g/L) of the drug in the ith release, V_i represents the volume of the release liquid at the ith sampling and MD means the mass of drug loaded (g).

Human bone mesenchymal stem cells (hMSCs) were originally purchased from Cell Bank of Shanghai Institutes for Biological Sciences. The PHBV and PP5 scaffold were sterilized by ultraviolet irradiation for 30 min and then placed in a 48 well cell culture plate. The hMSCs were seed on both materials at a density of 5 × 10⁴ cells/well, incubated at 37°C, 5% CO₂ atmosphere and 95% humidity for predetermined durations of 24 h, 48 h and 72 h. Then the materials with adhered cells were rinsed with PBS, stained with phalloidin and 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) for 30 s, and finally observed under inverted microscope.

In order to quantitatively evaluate the cell viability on both materials, CCK8 assay was used. Following incubation as described above, 10 ul CCK8 was added to each well and the plate was incubated in darkness at 37°C for 4 h, the absorbance (OD) values were measured at 450 nm (iMark, Bio-rad, USA).

The mRNA expression of collagen I (COL I), Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), osteoprotegerin (OPG) and alkaline phosphatase (ALP) were quantified to assess the osteogenic differentiation of different scaffolds by Real-Time PCR. hMSCs were adhered in 6-well plates and RNA were harvested after osteogenic induction for 7 days using TRIzol reagent (Invitrogen). Prime Script RT reagent kit (Takara, Shiga, Japan) was used for mRNA to be reversely transcribed into complementary DNA. ABI 7,900 was used for quantitative analysis of the reverse transcription reaction. The data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and analyzed by the 2^−ΔΔCT method.

Alizarin red S staining was used to evaluate the extracellular calcium deposition, revealing individual osteo-induction capacity of different scaffolds. HBMSCs were seeded into 24-transwell plates and co-cultured with PHBV or PP5 in osteogenic differentiation medium for 21 days. After 21 days, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min and then stained with Alizarin Red S (2% aqueous, Sigma) solution for 30 min.

Femur defect models were established on sixteen 8-week-old male SD rats to examine the repair efficacy of the PP5 scaffolds. The femoral defects model was prepared according to previously described protocols (Chou et al., 2008). The experimental rats underwent general anesthesia with intraperitoneal injection of 50 mg/kg pentobarbital sodium All animals were cared and experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011 revision). The experimental protocol was approved by the Ethics Committee of Affiliated Hospital of Guangdong Medical University (No. PJKT 2022-046). All possible efforts were made to reduce the number of rats used and discomfort to the rats. Afterwards, the surgical sites were shaved and disinfected. A bone defect of approximately 4 mm in diameter and 5 mm in depth was created at the right femoral epicondyle using a slow-speed cylindrical burr. Subsequently, according to the type of scaffolds implanted, the animals were randomly divided into two groups (8 rats per group): (1) no treatment (defect control), (2) PP5-scaffold. The post-operative pain was controlled by intra-muscular injection of 5 mg/kg morphine every 4 h. Two months post-implantation, the rats were sacrificed and the femurs were extracted for micro-CT analysis. 3D bone reconstruction and quantitative analysis of bone regeneration were conducted using the system software. Bone volume/tissue volume (BV/TV) and bone mineral density (BMD) of each sample were analyzed and calculated. Afterwards, the collected femur samples were decalcified and prepared for hematoxylin-eosin (H&E) staining to visualize bone regeneration.

Transwell system was used to analyze cell migration, cells were seeded in the top chambers of the transwell migration plates in DMEM containing 10% FBS. At 24 h after seeding, cells that migrated to the underside of the trans well were stained with 0.5% crystal violet for 5 min and imaged in ten random fields. To investigate the invasion and migration of cells in PP5 or MT-loaded PP5 scaffold, the cells (5 × 10⁵) were injected to the scaffold with a designated site, and the cell migration away from the injection sites was monitored with a light microscope (Leica, Germany) and quantified using ImageJ.

The protocols of use and care of the animals were reviewed and approved by Institutional Animal Care and Use Committee of Affiliated Hospital of Guangdong Medical University. 2.0 × 10⁵ LM-8 (murine osteosarcoma cells) per mouse were injected subcutaneously into the leg of the nude mouse (6 week-old Balb/c, Vital River Laboratory Animal Technology Co. Ltd., Beijing, China). When the volume of the tumor grew to about 300 mm³, the mice were randomly divided into three groups (n = 4). A small incision was made on the leg of the mouse and the functional cuboid scaffold (length: 8 mm, width: 2 mm, height: 2 mm) was implanted under the tumor. The first day of the treatment was regarded as Day 0. The weight of tumor was measured after 1 month. The tail vein injection was used for MT group treatment, and the dose of MT was consistent with that of MT + PP5 group.

Western blotting was used to determine the protein expression level of LC3 II/LC3 I and P62/GAPDH.The cellular proteins were extract with by cell lysis buffer (Invitrogen, United States). Protein samples were separated on 10% SDS-PAGE, followed by transferred onto PVDF membranes (Millipore, United States). The membranes were blocked with 2% bovine serum albumin (BSA) in PBST for 1 h at room temperature, then the membranes were incubated with the primary antibodies and secondary antibodies for 1 h at 37°C. Finally, the membrane was observed by sensitive enhanced chemiluminescence (ECL) detection kit (Beyotime, China) and bands were detected by ChemiDoc Imaging Systems (Bio-Rad, United States).

The immunocytochemistry was performed on cells grown on 22 × 22 mm coverslips. Wash the cells with PBS for 3 × 5 min. Fix the cells on a coverslip and fix them with 3.7% formaldehyde in PBS for 5 min. Permeabilize cells with PBS containing 0.1% Triton X-100 for 3 min. Block with 7.5% BSA in PBS. Incubate with primary antibodies diluted in PBS containing 0.5% BSA. Wash the cells 3 times. Incubate with secondary antibodies for 1 h. Counterstain the nuclei with PBS containing 10 ng/mL Hoechst 33342 for 3 min. Fluorescence is observed under a microscope.

Statistical data analysis was performed with SPSS software. Data from at least three independent experiments were collected, analyzed, and expressed as mean ± standard deviation (SD). Results with p-values of < 0.05 were considered statistically significant.

In order to overcome the limitations of PHBV, we opted to synthesize PP5 using PBHV and mPEG5k as the primary raw materials. The chemical structure of PP5 was confirmed through 1H NMR analysis as depicted in Figure 1, δ = 1.5−2.03, 2.32,4.0−4.19,4.32−4.47 ppm belongs to PHBV (a-g) while δ = 3.66 ppm was corresponded to mPEG5k, which confirmed that we graft mPEG5k to PHBV successfully (Park et al., 1998; Pasek-Allen et al., 2023). The structure of PEG5k, PHBV and PP5 were characterized by FT-IR (Figure 1). In the spectrum of mPEG5k, the absorption peak at approximately at 2,887 and 1,467 cm⁻¹ respectively are associated with the stretching and bending vibration of methylene (Xiang et al., 2013). And the peaks also appear in spectrum of PP5, therefore, the FT-IR results confirm that PP5 has been produced.

We utilized the material-cell co-culture method to evaluate the cytotoxicity of PBHV and PP5 and determine their biocompatibility. Human bone marrow mesenchymal cells were co-cultured with the materials for 24, 48, and 72 h, and cell viability was assessed using the CCK8 assay kit. Our findings indicated that both materials displayed no-cytotoxicity and showed some pro-proliferative effect. Moreover, PP5 exhibited a more pronounced pro-proliferative effect compared to PHBV (Figure 2C). Cellular immunofluorescence staining for cytoskeletal proteins (β-tublin) was performed to evaluate cell spreading on the scaffold materials. Fluorescence analysis (Figure 2A) demonstrated that cells exhibited greater extensibility on PP5. In conclusion, the addition with mPEG5k resulted in the synthesis of PP5 biomaterials with improved hydrophilicity and biocompatibility.

To further evaluate the osteogenic ability of PP5, we conducted both cellular and animal models to investigate the osteogenic effect of each material. We co-cultured both materials with cells and performed RT-PCR analysis of osteogenesis-related genes. (Figure 3A). The expression of osteogenesis-related genes such as ALP, OPG, OCN, Runx2, and Collagen I were significantly higher in the PP5 group compared to the PBHV group. In the alizarin red mineralized nodules staining assay, the PP5 group exhibited higher mineralized nodules compared with control group. For the in vivo study, we utilized a femoral defect model in SD rats, a small bone defect was artificially created in the lower femur of SD rats and then implanted with PBHV and PP5, respectively. After euthanasia at 8 weeks postoperatively, the specimens were taken for Micro-CT analysis and HE. As shown in Figure 4A, after data analysis by specific software, the bone mineral density (BMD), bone volume fraction (BV/TV), bone trabecular number (Tb.N), and bone trabecular thickness (Tb.Th) were found to be statistically higher in the PP5 group compared to the PBHV group (p < 0.05). These differences were also observed in HE staining (Figure 4B). From the above, it can be concluded that PP5 has a significantly better bone healing promoting effect than PBHV.

As a potential drug against bone tumors, we proposed to construct a local drug delivery system by loading melatonin to provide a new option for improving the quality of comprehensive management of bone tumors after surgery. Through GelMA, we successfully loaded melatonin on PP5 scaffold and performed release experiments. (Figure 5). The loading capacity increased with decreasing gel concentration, and the loading efficiency initially increased with the increasing concentration and reached a peak at the gel concentration of 1%, so the 1% gel concentration was used for subsequent experiments. In the drug release curve, the melatonin release process was able to sustain the release for more than 14 days without sudden mass release, and the release efficiency of MT was able to reach more than 97.1%. The result indicating that the MT loaded PP5 has the ability to achieve sustained and slow release of MT.

To evaluate the anti-tumor efficacy of the drug delivery system, we conducted both in vitro and in vivo experiments. Specifically, we treated tumor cells (MG-3) with two different scaffold and melatonin for a period of 24 h. We then performed cell transwell assays and cell proliferation assays (Figures 6A–C). Both the free melatonin group and MT + PP5 group exhibited anti-bone tumor abilities. In addition, the cell proliferation assay measured by the CCK8 kit also demonstrated inhibitory effects of the above two groups on the proliferation of MG63 cells. Interestingly, the MT + PP5 group showed stronger anti-tumor ability than the free MT group in both experiments.

In vivo experiments, we utilized a nude mouse allogeneic subcutaneous tumorigenesis model, once the subcutaneous tumor volume reached 300 mm² in nude mice, we implanted scaffold materials subcutaneously in the tumors, and the tumor specimens were euthanized and weighed after 14 days (Figure 6D). The tumors in the MT + PP5 group were significantly smaller than those in the other three groups.

To further explore the enhanced antitumor effect of melatonin, we examined the autophagic effect of melatonin on tumors. Western blotting and cellular immunofluorescence showed differential expression of markers associated with cellular autophagy (Figure 7A). The LC3B protein expression was upregulated in the MT group and MT + PP5 group, while P62 protein expression was decreased, with a greater difference observed in the MT + PP5 group. The difference in protein expressions was confirmed through cellular immunofluorescence (Figure 7B). Based on the results, it can be inferred that PP5 can enhance the anti-tumor effects of melatonin by promoting autophagy.