Skip to main content
Download PDF

Enhanced effects of slowly co-released TGF-β3 and BMP-2 from biomimetic calcium phosphate-coated silk fibroin scaffolds in the repair of osteochondral defects

Journal of Nanobiotechnology volume 22, Article number: 453 (2024) Cite this article

A Correction to this article was published on 27 January 2025

This article has been updated

Abstract

Bioactive agents have demonstrated regenerative potential for cell-free bone tissue engineering. Nevertheless, certain challenges persist, including ineffective delivery methods and confined therapeutic potency. Here, we demonstrated that the biomimetic calcium phosphate coating system (BioCaP) could effectively uptake and slowly release the incorporated bioactive agents compared to the surface absorption system via osteoclast-mediated degradation of BioCaP coatings. The release kinetics were determined as a function of time. The release rate was stable without remarkable burst release during the first 1 day, followed by a sustained release from day 7 to day 19. Then, we developed the bi-functional BioCaP-coated silk fibroin scaffolds enabling the effective co-delivery of TGF-β3 and BMP-2 (SFI-T/SFI-B) and the corresponding slow release of TGF-β3 and BMP-2 exhibited superior potential in promoting chondrogenesis and osteogenesis without impairing cell vitality in vitro. The SFI-T/SFI-B scaffolds could improve cartilage and bone regeneration in 5 × 4 mm rabbit osteochondral (OC) defect. These findings indicate that the biomimetic calcium-phosphate coated silk fibroin scaffolds with slowly co-released TGF-β3 and BMP-2 effectively promote the repair of OC defects, hence facilitating the future clinical translation of controlled drug delivery in tissue engineering.

Graphic Abstract

Introduction

Articular injuries caused by trauma, inflammations, stress, and aging [1, 2] are common clinical conditions. These injuries are graded by lesion extent, depth and severity. Osteochondral (OC) defects are classified as the most severe and debilitating types, involving complete cartilage loss and partial damage of the beneath subchondral bone. OC defects can be detected in up to 61% of all knee injuries [3] and are disreputable for poor ability to fully self-repair, especially for significant OC defects, thus surgery being imperative. However, current surgical strategies such as arthroscopic treatment, microfracture, articular drilling, and autologous allograft transplantations are still trapped by problems of fibrocartilage healing, donor site morbidity, disease transmission, and additional economic burden [2,3,4,5,6].

These limitations encourage tissue engineering approaches to focus on restoring or replacing the damaged OC units. Cell-free tissue engineering techniques have attracted much more attention to avoid cell-derived adverse immune responses [6], possible tumorigenicity [7,8,9], and expensive culture costs [9]. This further highlights the importance of the cell-free tissue engineering system to bear the biological cues, which are used to provide by the host cells, to induce chondrogenesis and osteogenesis. Two powerful bioactive agents are transforming growth factor beta 3 (TGF-β3) and bone morphogenetic protein 2 (BMP-2) [10]. They are both members of the TGF-β family, which is reported to respectively target chondrocytes and osteoblasts and inhibit the in-growth of other cells (e.g., fibroblast) [11]. TGF-β3 has been confirmed as a mesenchymal stem cell (MSC) homing molecule [12]. Thus, endogenous MSCs can be recruited into the cell-free scaffolds and then participate in the following tissue engineering in the presence of TGF-β3 [12,13,14,15]. Meanwhile, BMP-2 is a well-recognized potent osteo-inducer required to initiate bone repair [16]. BMP-2 has also been reported to decrease potential fibrosis while generating maximal newly formed bones, improving the quality of bone tissue engineering. However, the clinical promotions of TGF-β3 and BMP-2 are limited due to some unmet problems. Firstly, these bioactive agents are loaded onto the surface of scaffold materials where they are loosely absorbed and rapidly diffused into the local milieu, failing to maintain a long-lasting valid concentration in defect areas. Secondly, TGF-β3 and BMP-2 are prone to be inactivated by enzymes and non-physiological temperature and pH. Therefore, direct surface absorption of TGF-β3 and BMP-2 onto scaffolds cannot effectively obtain tissue restoration.

Scaffolds are essential to bone tissue engineering, providing cell adhesion 3D structure and mechanical support in OC defect areas. Our studies chose silk fibroin (SF) based on its exceptional biocompatibility, adherence, and biodegradability [5, 17, 18]. Being more substantial and more durable than other natural materials, SF has been widely used in skeletal tissue engineering, such as bone and cartilage [18]. Besides, the SF heavy chains contain plenty of glycine (45.9%) and alanine (30.3%), which can serve as templates for synthesizing required proteins in defect areas [18]. Kim and other researchers have reported that SF can suppress inflammatory responses by reducing related cytokines [18,19,20], which is beneficial for SF in modulating the initial inflammatory reactions when implanted [21]. However, the inferior compression performance [22] limited the promotion of SF as an ideal biomaterial to regenerate OC defects, simultaneously involve bone and cartilage restoration. Modified materials based on SF with higher compressive modulus and superior biocompatibility, chondrogenesis and osteogenesis were needed.

Biomimetic calcium phosphate (BioCaP) coatings comprised of octacalcium phosphates (OCP) and amorphous calcium phosphates (ACP) have been developed and used to delivery osteogenic factors in cranial bone tissues regeneration [23] or surface modification of dental implants [24] since introduced by Kokubo et al. in 1990 [25, 26]. Different from other coating techniques (e.g., plasma spraying, coating via sol–gel phase shifting, and magnetron sputtering), the BioCaP coatings are synthesized under simulated physiological conditions (T ≈ 37 °C and pH ≈ 7.4), endowing the BioCaP coatings a prominent advantage that the bioactivities of proteins can be intactly retained when incorporated into the OCP layers. Previous studies have confirmed that the proteins were incorporated into the OCP layers via a strong covalent bond [27] instead of loosely connected via a surface absorption mode. In that case, the incorporated proteins can be protected from being inactivated and released slowly along with the degradation of BioCaP coatings.

Herein, this study further explored the application field of the BioCaP coating, which was used to deliver the chondrogenic TGF-β3 at the first time. Besides, the slow-release of BMP-2 via the BioCaP coating was also firstly applied in reconstruction of osteochondral. In this study, the bi-functional SF scaffold (SFI-T/SFI-B) was designed, from which TGF-β3 and BMP-2 are slowly co-released via the BioCaP coating, to regenerate cartilage and bone hierarchically at the same time. Endogenous MSCs are expected to be recruited into SF scaffolds when TGF-β3 is released. The long-lasting and slowly co-released TGF-β3 and BMP-2 from SFI-T/SFI-B could persistently induce chondrogenesis and osteogenesis on the temples of SF. In vitro physicochemical characters, recruitment effects, biocompatibility, chondrogenesis, and osteogenesis of different scaffolds were evaluated. The in vivo therapeutic effects were tested via a rabbit OC defect model (Fig. 1).

Fig. 1
figure 1

Schematic illustration of the fabrication and application of SFI-T/SFI-B scaffolds. a The in vitro fabrication process of SFI-T/SFI-B scaffolds. b With the slowly co-released TGF-β3 and BMP-2 from SFI-T/SFI-B scaffolds, enhanced chondrogenesis and osteogenesis of host BMSCs, cartilage repair, and bone repair could be finally obtained in vivo

Full size image

Material and methods

Fabrication of silk fibroin scaffolds

Aqueous silk fibroin solution (2 wt%) was prepared following the previously reported protocol [13]. Briefly, bombyx mori cocoons were cut into small pieces and degummed with boiled 0.02 M sodium carbonate (Na2CO3, 223530, Sigma-Aldrich, USA) solution for 1 h to obtain the silk fibers. Then the obtained fibers were washed with deionized water three times and dissolved in 9.3 M lithium bromide solution (229733, Sigma-Aldrich, USA) at 60 °C for 4 h before dialyzing against deionized water for 3 days. Thus, the silk fibroin solution was cast in an appropriate mold (5 mm diameter × 3 mm thickness and 5 mm diameter × 1 mm thickness), frozen at − 20 °C, and subsequently lyophilized. The transition from random coils to β-sheet structures of the fabricated silk fibroin scaffolds was induced with 100% percentage ethanol, and the scaffolds acquired insolubility in aqueous solutions.

Biomimetic calcium phosphate coating procedure

Prior to coating, the SF scaffolds were sterilized by being immersed in ethanol (70% v/v) with radiation (UV) for 30 min, followed by rinsing with sterile phosphate buffer (PBS, 20012027, Gibco, USA) more than three times to remove the traces of ethanol [28]. Then SF scaffolds were coated with a layer of BioCaP according to the well-established protocol refined by our group [27, 29]. Initially, scaffolds were immersed in the 5-times-concentrated simulated body fluid (681.5 mM NaCl (S9888, Sigma-Aldrich, USA), 12.5 mM CaCl2•2H2O (223506, Sigma-Aldrich, USA), 5.0 mM Na2HPO4•2H2O (71643, Sigma-Aldrich, USA), 21.0 mM Na2CO3 (223530, Sigma-Aldrich, USA), 7.5 mM MgCl2•6H2O (M9272, Sigma-Aldrich, USA)) at 37 °C for 24 h. Thus, a thin (1–3 μm) and dense layer of ACP was formed on the surface of the SF scaffold. The OCP layer was deposited on the surface of the ACP layer by immersing the samples (10 ml per sample) for 48 h at 37 °C in a supersaturated solution of calcium phosphate (4.0 mM CaCl2•2H2O, 136.5 mM NaCl, 2.0 mM Na2HPO4•2H2O), buffered with 50 mM TRIS (pH 7.4, 252859, Sigma-Aldrich, USA)). SF coated with BioCaP coatings was recorded as SFI. The whole coating process was performed under sterile conditions.

Applications of BMP-2 and TGF-β3

Surface absorption of BMP-2 and TGF-β3

The SF scaffold was immersed in TGF-β3 (bs-47205P Bioss, China) aqueous solution (1 µg/ml of phosphate-buffered saline, pH 7.4, 10 ml per scaffold) at 37 ℃ for 48 h and the adsorbed depot of TGF-β3 was produced on the surface of the SF scaffold, recorded as SFA-T scaffold. The SF scaffold was immersed in BMP-2 (bs-0514P, Bioss, China) aqueous solution (1 µg/ml of phosphate-buffered saline, pH 7.4, 10 ml per scaffold) at 37 ℃ for 48 h and the adsorbed depot of BMP-2 was produced on the surface of the SF scaffold, recorded as SFA-B scaffold. The whole process was performed under sterile conditions.

Biomimetic coatings incorporated BMP-2 and TGF-β3

As described above, SF was first coated with an ACP layer after 24 h of reaction. TGF-β3 was added into the OCP solution as the working solution for the following coating process at a final 1 μg/ml concentration. SF coated with ACP layer was then immersed in the TGF-β3 working solution for 48 h at 37 °C to simulate the physical condition, recorded as the SFI-T scaffold. Similarly, BMP-2 was added into the OCP solution as the working solution for the following coating process at a final 1 μg/ml concentration. Another SF coated with ACP layer was then immersed in the BMP-2 working solution for 48 h at 37 °C, recorded as the SFI-B scaffold. After the deposition process, the biomimetic coatings incorporated BMP-2 or TGF-β3 were obtained. The whole coating process was performed under sterile conditions.

Scanning electron microscopy (SEM)

The surface structural characterization and porous structure of scaffolds were accomplished by SEM (S-3400N, Hitachi, Japan) after gold-sputtered.

Fourier transform infra-red spectroscopy (FTIR)

The conformation of different scaffolds was evaluated by attenuated total reflectance (ATR) model in the FTIR instrument (IS5, Thermo Fisher Scientific, USA) after being mixed with potassium bromide.

Compression modulus of different SF scaffolds

The compressive test (wet state) of different scaffolds was performed with a Universal Testing Machine (Instron3366, Instron, USA) under a 1kN load cell at room temperature. The speed was set at 2 mm/min until there was a 70% reduction in sample height.

Swelling ratio of different SF scaffolds

The swelling ration of the SF scaffold was estimated according to the gravimetric method [5]. Before the swelling ratio test, all scaffolds were dried with the fume hood for 24 h. The dried scaffolds (M0) were immersed in PBS at 37 °C. After different immersion intervals (5, 10, 20, 30, 40, 60, 80, 100 and 120 min), the wet scaffolds were weighted (M1) after the excess water on the surfaces was cleared away with filter paper. The swelling ration of the scaffold was calculated as follows:

$$\text{Swelling ration } = \text{(M1-M0)}/{\text{M0}}$$

Cell-mediated protein release kinetics

Bovine serum albumin labelled with Alexa Fluor 488 (Alexa-BSA, bs-0292P-AF488, Bioss, China) was adopted as a model protein to demonstrate protein release kinetics. As described above, Alexa-BSAs were added to the OCP solution at a final concentration of 0.5 μg/ml to be incorporated in the BioCaP coating on the surface of the SF scaffold, recorded as SFI-BSA. The SF scaffolds were immersed in the Alexa-BSA solution (0.5 µg/ml, 10 ml for per scaffold) at 37 ℃ for 48 h to establish surface absorption of Alexa-BSA (SFA-BSA).

According to previous studies, the BioCaP coating can be digested by osteoclasts [30, 31]. Thus, the passive and cell-mediated release of Alexa-BSA tested over 19 days as, reported in these studies [30, 31]. 1 \(\times\) 106 C57BL/6 bone marrow-derived monocytes (BMDMs) were seeded on different SF scaffolds and cultured with the complete α-MEM medium (12571063, Gibco, USA) with 10% fetal bovine serum (30044333, Gibco, USA) and 1% penicillin–streptomycin (SV30010, Hyclone, USA), with/without the stimulation of RANKL (50 ng/ml, 315–11, PeproTech, USA) and M-CSF (30 ng/ml, 315–02, PeproTech, USA) to investigate the cell-mediated release of Alexa-BSAs. SF scaffolds without BMDMs were incubated in a complete α-MEM medium to investigate the passive release of Alexa-BSAs. The culture medium was collected and changed every 3 days. The collected medium (n = 4 per time point) was tested with fluorescence spectroscopy (SpectraMAXM3, Molecular Devices, USA), using 488 nm as the excitation spectrum and 519 nm as the emission spectrum. The fluorescence readings were converted into the release amount of protein with the standard curve generated from a dilution series of Alexa-BSA.

After 19 days of experiments, the residual Alexa-BSA in SF scaffolds was checked by immersing the scaffolds in 10% EDTA-2Na solutions (BioFroxx, 1108GR500, China).

$${\text{Uptake}}\,\,{\text{efficiency}}\,\,{ = }\,\, \left( {{\text{released}}\,\,{\text{and}}\,\,{\text{residual}}\,\,{\text{BSA}}} \right) \,\, \div \,\,\left( {{\text{BSA}}\,\,{\text{added}}\,\,{\text{into}}\,\,{\text{the}}\,\,{\text{OCP}}\,\,{\text{solution}}} \right)\, \times \,{1}00\%$$
$${\text{Utilization}}\,{\text{ ration}}\,\,{ = } \left( {{\text{released}}\,{\text{ BSA}}} \right) \, \div \,\left( {{\text{BSA}}\,{\text{ added}}\,{\text{ into}}\,{\text{ the}}\,{\text{ OCP}}\,{\text{ solution}}} \right)\, \times \,{1}00\%$$

Wound healing assay

The wound-healing assay assessed the effect of BMP-2 and TGF-β3 on cell migration. Bone marrow stem cells (BMSCs) were cultured in a 6-well plate until 80% confluence was reached. Before scratching, BMSCs were synchronized through serum starvation for 24 h. The single cell layer was gently scratched with a new sterilized 200 µl pipette tip in each well. The well was rinsed twice with sterilized PBS to remove the cell debris and cultured with fresh culture medium containing BMP-2 (0 µg/ml, 0.3 µg/ml, and 1 µ/ml) or TGF-β3 (0 µg/ml, 0.3 µg/ml, and 1 µg/ml). 24 h later, the cells were photographed and the gap area was quantitatively evaluated using Image J software.

Cell seeding

Prior to seeding, SF scaffolds of different groups were incubated with cell culture media overnight (37 °C, 5% CO2). After incubation, the SF scaffolds were taken out for dehydration; more than 4 h were required. Cells with the required amount were seeded onto each dehydrated SF scaffold and cultured without disturbance for cell infiltration (37 °C, 5%CO2). After 30 min, the cell medium was gently added at the corner of each well without disturbing the cellscaffold complex. The culture media was refreshed at 3-day intervals during the entire experiment.

Bioactivity and biocompatibility assessments

To demonstrate BioCaP coating process did not impair the bioactivity of incorporated protein, 1 \(\times\) 106 BMSCs at passage 3 were seeded onto SF, SFI, SFA-T, and SFI-T scaffolds. The cell-scaffold complexes were fixed with 4% paraformaldehyde after 48 h incubation. The expression of Col-II (ab185430, Abcam, USA) was analyzed using confocal laser scanning microscopy (CLSM). ImageJ software was used to quantify the expression.

Considering the silk fibroin itself can also been stained with dye, which may affect the observation of cell morphology [32], we detect the cell proliferation ability with cck-8 assay and cell flow assay to check the biocompatibility of scaffolds. The cell-scaffold complex was also incubated with the cell counting kit working solution of a cell counting kit (CCK8, CK04, DOJINDO, Japan) at 37 °C for 1 h protected from light in different time intervals to detect cell proliferation. After incubation, the optical density (OD) was measured with the multi-plate reader (SpectraMax M3, Molecular Devices, USA) at a wavelength of 450 nm. In order to evaluate the biotoxicity of different groups, scaffolds were saturated with α-MEM for 24 h at 37 °C. The supernatant was filtered through 0.22 µm filters and used to incubate with BMSCs. 24 h later, the cells were harvested and stained via Annexin V-FITC/PI Apoptosis Detection Kit (A211, Vazyme, China). Stained cells were analyzed through the flow cytometer within 1 h (FACSCalibur, BD).

RNA isolation and reverse transcription-quantitative polymerase reaction (RT-qPCR)

Functional changes of the cell morphology will regulate the genes expression. Therefore, we check the expression of chondrogenesis and osteogenesis genes for different scaffolds. MC3T3 and ADTC5 cell lines were used to evaluate the osteogenesis and chondrogenesis related effects of different groups. The RNA was extracted using an RNA Extraction Kit (0026, Beyotime, China) and transcribed with a cDNA Reverse Transcription kit (R223, Vazyme, China). A quantitative polymerase reaction was performed using SYBR Green Mix (1725124, BIO-RAD, USA). The RNA expressions were examined for the following factors: Col-II, SOX-9, Col-I, and OCN. GAPDH was used as the internal reference and the primer sequences are provided in supplementary data Table S1.

In vivo investigation

Twenty-eight adult male New Zealand white rabbbits (~ 2.0 kg) were adopted to evaluate the in vivo efficacy of different scaffolds which was approved by the Ethical Committee of Nanjing Agriculture University (PT2021003). All animal study procedures were performed according to the ethical rules and regulations of Nanjing Agriculture University.

Animal model

Twenty-eight rabbits were used in this study, four of which used in the preliminary experiment to make sure the knee OC defects model (diameter: 5.0 mm, depth: 4.0 mm, Supplementary data Fig S1) can be established. After that, twenty-four rabbits (forty-eight defects) were divided into 6 groups listed in Table 1 (n = 4 defects per group and per timepoint). The upper 1/4 layer and lower 3/4 Layer of each group were separately fabricated according to the methods described in “2.3 Applications of BMP-2 and TGF-β3” and then glued together as one bilayer scaffold using fibrin glue [32]. Then The bilayer scaffold was implant in the defect site and TGF-β3 and BMP-2 can be co-released spatially.

Table 1 Silk fibroin scaffold groups information for in vivo experiments
Full size table

All rabbits were anesthetized by intravenous injection of 0.3 ml/kg Zoletil 50 (Virbac, China) before surgery. The region of operation was shaved and aseptically prepared for operation. The knee OC defects were established on the position of the trochlear groove. After the implantation of scaffolds, the wounds were sutured, and penicillin (030011253, Hebei Yuanzheng Pharmaceutical Co.,Ltd., China) was intramuscularly injected into each rabbit for 7 days following the surgery.

Micro-CT assessment

The knee joint samples were collected 5 and 10 weeks after the surgery. Before paraffin embedding, samples were assessed by a Micro-CT scanner (Skyscan 1176, Bruker). The osteogenesis properties, more specifically, the bone volume per total sample volume (BV/TV), the mean trabecular number (Tb. N), and the mean trabecular separation (Tb. Sp) were calculated.

Different combination of layered scaffold with/without growth factors and BioCaP coatings was used to repair osteochondral defects in rabbit femoral grooves.

Macroscopic and histological examination

Samples from each group were photographed and examined according to the International Cartilage Repair Society (ICRS) macroscopic assessment scale. After gross examination, samples were fixed in 4% formalin, decalcified in 10% EDTA for 2 months, and then embedded in paraffin. Sections from each sample were stained with H&E (G1005, Servicebio, China), Safranin O & Fast Green (G1371, Solarbio, China), Goldner stain (G3550, Solarbio, China) and immunohistochemistry of Col-II (ab185430, Abcam, USA) and Runx2 (bs-1134R, Bioss, China).

Results

Fabrication and characterization of different SF scaffolds

The scheme in Fig. 1a illustrates the fabrication and structure of the BioCaP-coated SF scaffolds. The ACP layer was deposited on the surface of the porous SF scaffold as the seeding layer for the subsequent deposit of the OCP layer. BMP-2 or TGF-β3 were added to the supersaturated CaP solution to be incorporated into the BioCaP coating. The experimental groups in vitro were listed in Table 2.

Table 2 In vitro experimental groups
Full size table

TGF-β3 was adopted as the model protein to demonstrated different scaffolds’ characters because of the similar structures between TGF-β3 and BMP-2. As shown in Fig. 2a, pure SF scaffolds(i) and SFA-T scaffolds (ii) demonstrated a similar smooth porous structure. Coated with BioCaP coating, the surfaces of SFI scaffolds displayed a plate-like crystal structure (iii-iv). Incorporation of TGF-β3 in SFI-T scaffolds led to a slightly bent and more compact crystal without changing the fundamental structures of BioCaP coatings (v, vi). The FTIR spectra (Fig. 2b) revealed the silk-II conformation in silk fibroin of different scaffolds. Peaks at 1705 cm−1 and 1620 cm−1 attributed to silk-II were observed among SF, SFA-T, SFI, and SFI-T scaffolds. The peaks at 1022, 602, and 557 cm−1 are attributed to the PO43− bond that appeared in SFI and SFI-T scaffolds due to the deposition of BioCaP coatings. Moreover, the presence of BioCaP coatings led to a decreased swelling ratio and porosity but increased compressive modulus in SFI and SFI-T scaffolds, as indicated in Fig. 2e, f. The swelling ratios for SF, SFA-T, SFI, and SFI-T scaffolds were 11.41, 11.36, 9.94, and 9.61. The comparative lower swelling ratio for SFI-T scaffolds indicated their higher stability. The porosity for SF, SFA-T, SFI, and SFI-T scaffolds were 64.8, 63.3, 51.7, and 50.9%. The compressive modulus SF, SFA-T, SFI, and SFI-T scaffolds were 88.96, 80.51, 147.59, and 211.34 kPa, indicating the application of inorganic BioCaP coating enhanced the mechanical strength. The SFI-T scaffolds displayed the highest Col-II expression (Fig. 2f, g), about a 1.8-fold increase in fluorescent intensity compared to SF scaffolds.

Fig. 2
figure 2

Physicochemical characterizations of different SF scaffolds. a Scanning electron microscopy of SF(i), SFA-T(ii), SFI (iii-iv) and SFI-T(v-vi) scaffolds. b Fourier transform infraed spectroscopy spectra of SF, SFA-T, SFI and SFI-T scaffolds. c–e The swelling ratio, porosity, and compressive modulus of SF, SFA-T, SFI and SFI-T scaffolds. f The representative images of Col-II expression of BMSCs seeded on scaffolds with fluorescent dyes after 48 h incubation. Scale bar = 10 µm. g Quantity of Col-II expression. *P < 0.05, **P < 0.01

Full size image

Cell-medicated slow release of BSAs from BioCaP@SF scaffolds

Previous studies have reported the proteins incorporated into the BioCaP coating can be released when the coating is digested. The basic passive release profile and the cell-medicated release profile were monitored. Scaffolds incubated with culture media were aimed to monitor the passive release of Alexa-BSAs. Scaffolds seeded with BMDM were aimed to monitor the cell-mediated release of Alexa-BSAs. Besides, BMDMs were induced as osteoclasts under the stimulation of CSF and RANKL (Fig. 3b). As shown in Fig. 3c, Alexa-BSAs were successfully loaded onto SF scaffolds. More BSAs were up-taken in SFI-BSA scaffolds compared to SFA-BSA scaffolds on day 0 under the same reaction conditions. The uptake efficiency for SFI-BSA scaffolds was 30.08 ± 7.11%, almost sevenfold higher than SFA-BSA scaffolds (Fig. 3g).

Fig. 3
figure 3

Release kinetics of BSAs from SFA-BSA and SFI-BSA scaffolds. a Schematic illustration of the process for BSA incorporation and cell-mediated release. b TRAP stain. BMDMs were harvested and induced into osteoclasts with RANKL (50 ng/ml) and CSF (30 ng/ml). White arrows indicated the osteoclasts. Scale bar = 100 µm. c, d The visualized uptake and residual protein of SFA-BSA and SFI-BSA scaffolds through CLSM. e, f Release profiles of SFA-BSA and SFI-BSA scaffolds over 19 days. g The BSA uptake efficiency and final utilization ratio via surface absorption (SFA-BSA) and BioCaP coated (SFI-BSA) modes. *P < 0.05

Full size image

Throughout 19 days of monitoring, most of the BSAs loaded in SFI-BSA scaffolds of culture medium group and BMDM group were not released (Fig. 3d). Most of the BSAs loaded in SFI-BSA scaffolds were released in (BMDM + CSF, RANKL) group, in which osteoclasts were induced (Fig. 3d). The release kinetics in Fig. 3e and Fig. 3f can further confirm the above observations. Among 3 SFA-BSA groups, BSAs were almost rapidly released within 1 day (Fig. 3e). BSAs were slowly released in the presence of osteoclasts, and the amount of released BSAs was significantly higher than the other two groups. The cell-mediated release kinetics of BSAs from SFI-BSA groups were determined as a function of time. The release of BSAs was stable without remarkable burst release during the first 1 day, followed by a sustained higher release from day 7 to day 19 (Fig. 3f). After 19 days’ observation, 66% of BSAs from SFI-BSA scaffold exposed to osteoclasts was released, and the final utilization ratio was 20.53 ± 1.73%, almost fivefold higher than SFA-BSA groups.

Cell recruitment, biocompatibility, chondrogenesis and osteogenesis properties of BioCaP@SF scaffolds

The wound healing assay showed that BMSCs migrated faster under the stimulations of TGF-β3 and BMP-2 (Fig. 4a) and the migration rate was independent of their concentrations (Fig. 4b). The biocompatibility of biomaterials is the critical feature for their in vivo application. The results of the CCK8 assay for accessing the proliferations of BMSCs are shown in Fig. 4c. The results showed that the OD values increased from day 1 to day 7 for SF, SFI, SFI-T, and SFI-B scaffolds. Besides, the BMSCs incubated with different scaffold supernatants also displayed no significant difference in cell vitality (Fig. 4d, e).

Fig. 4
figure 4

Biocompatibility of different SF scaffolds. a BMSCs migration exposed to different concentrations of TGF-β3 and BMP-2 examined by the in vitro wound healing assay. The red lines indicate cell migration fronts. b Quantification of the migration rate. c Proliferation of BMSCs seeded on different scaffolds examined with CCK-8 at 1, 3, and 5 days. d A flow cytometer measured the live cell rate of BMSCs cultured with a relative scaffold supernatant. e Quantification of the live cell rate. f The diagram of cell culture experiments. g–j In vitro chondrogenesis and osteogenesis of different scaffolds. *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

As demonstrated in Fig. 4f, BMDM induced with CSF and RANKL were seeded on scaffolds and cultured in the upper chamber. ADTC5 (chondrocyte cell line) or MC3T3 cells (osteocyte cell line) were cultured in the lower chamber. The cartilage (Col-II and SOX9) and bone formation-related gene (Col-I and OCN) expression levels were evaluated (Fig. 4g–j). The above release kinetics showed proteins were almost entirely released within 1 day in the surface absorption group. In contrast, more proteins were detected persistently from day 7 to day 19 in BioCaP coated groups (Fig. 3e, f). The expressions of marker genes were in consistent with the trend of the above release kinetics. The surface absorbed TGF-β3 and BMP-2 were almost released within 1 day and enhanced the expressions of related genes at day 1(1.6-fold for Col-II, 2.1-fold for SOX9, 1.3-fold for Col-I, and 1.7-fold for OCN). Then, the expressions descended to a level similar to pure SF scaffolds from day 4 to day 10. The SFI-T or SFI-B scaffolds can persistently stimulate the expression of related genes since day 7, when TGF-β3 or BMP-2 began to be gradually released. After 10 days of experiments, the expressions of related genes in BioCaP@SF scaffolds incorporated with TGF-β3 or BMP-2 were the highest among groups (2.1-fold for Col-II, 4.1-fold for SOX9, 6.6-fold for Col-I, and 5.6-fold for OCN).

SFI-T/SFI-B scaffolds enhanced cartilage and bone repair in rabbit OC defects

In vivo investigation was carried out to explore the cartilage repair and subchondral bone reconstruction for the regenerative potential of scaffolds. Full-thickness osteochondral (OC) defects (diameter 5 mm, depth 4 mm) on the bilateral knee joints of rabbits were used to evaluate the regeneration ability of scaffolds for the chondral and subchondral bone layer.

The slowly co-released TGF-β3 and BMP-2 from the SFI-T/SFI-B scaffold continuously promoted the repair of OC defects compared to surface-absorbed TGF-β3 and BMP-2

To investigate the regeneration capacity of TGF-β3 and BMP-2 with different release modes, the OC defects in rabbits were treated (1) blank (Control group), (2) silk fibroin (SF group), (3) surface absorbed TGF-β3 and BMP-2 (SFA-T/SFA-B group), and (4) slowly co-released TGF-β3 and BMP-2 (SFI-T/SFI-B group). After the surgery, no adverse events related to scaffold implantation occurred throughout the study. The samples were harvested at 5 weeks or 10 weeks post-surgery. After that, the gross and micro-CT evaluation and histological staining analyses were carried out to assess the reconstruction of OC tissues.

Gross evaluation

Grossly, no regenerated tissues were detected and surrounding tissues collapsed in the blank Control group 5 weeks post-surgery (Fig. 5a). The SF and SFA-T/SFA-B group displayed a large amount of non-degraded scaffold and little reddish tissue covering on the cartilage defect, showing comparatively limited chondrogenesis of the SF scaffold, and the absorbed TGF-β3 in vivo. Compared to the 5-week samples, the 10-week samples of all experimental groups displayed a larger covered area of the cartilage-like tissue, showing the progression of cartilage repair (Fig. 5a). The slowly co-released experimental group (SFI-T/SFI-B) was repaired with smooth, well-integrated tissue. It was refining into the hyaline cartilage-like tissue, which was not observed in the other groups. Defects in the blank control group and surface absorption group (SFA-T/SFA-B) were still left with large holes in the center region, and only a little tissue formed on the marginal of defects. ICRS was adopted to evaluate all samples, and the results showed that the total scores of the regenerated cartilage tissue in OC defects increased after 5 and 10 weeks (Fig. 5e). The scores of blank Control group (5.25 ± 1.32 for 10 weeks, 2.88 ± 1.45 for 5 weeks) groups were lower than the other group, confirming the limited self-repair ability of cartilage. The scores of SFA-T/SFA-B groups barely increased from 5 weeks (6.08 ± 0.76) to 10 weeks (6.80 ± 1.60). While in SFI-T/SFI-B groups, the scores increased from 5 weeks (6.20 ± 1.52) to 10 weeks (10.00 ± 1.00), exhibiting long-lasting effects to support cartilage regeneration.

Fig. 5
figure 5

The slowly co-released TGF-β3 and BMP-2 from SFI-T/SFI-B scaffolds continuously promoted cartilage repair compared to surface-absorbed TGF-β3 and BMP-2. a Photographs of OC defects in the blank Control group and different scaffold groups. The red circle indicated the border of OC defect and inside was the regenerated tissue. b Histological H&E, c Safranin O & Fast Green, and d Col-II immunochemistry stain of OC defects. The red arrow in b indicated the border of the defect. The Brown stain in d indicated the positive stain. e Quantitative ICRS scoring analysis (n = 4). f Quantitative analysis for Safranin O & Fast Green stain (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Full size image
Micro-CT analyses

A 4 mm depth OC defect in rabbits involves the whole layer of cartilage and part of the subchondral bone. It was quite straightforward to distinguish the stage of bone repair using 2D reconstruction pictures from distinct cross-section views (Fig. 6a–d). To begin, the neo-bone formed from the edges and bottoms of the defects toward the center. The reconstructed images show that new subchondral bones filled the defect of SFI-T/SFI-B groups after 5 weeks of implantation, while the SFA-T/SFA-B groups showed significantly delayed bone formation. After 10 weeks, the regeneration process was at a similar lower level in the blank Control, SF/SF, and SFA-T/SFA-B groups; the SFI-T/SFI-B groups showed enhanced new subchondral bones formation. Quantitatively, the analysis of BV/TV, Tb.N, and Tb.Sp based on Micro-CT data (Fig. 6i–k and m–o) further revealed the different osteogenesis efficacy of different scaffolds. After 5 weeks, the BV/TV for blank Control and SF/SF groups were 8.3 ± 1.11 and 7.05 ± 1.97, respectively. The SFI-T/SFI-B groups showed the highest BV/TV (12.4 ± 1.27), almost 3 folds of the SFA-T/SFA-B groups (3.93 ± 1.44). Notably, the SFI-T/SFI-B groups at 10 weeks demonstrated the highest osteogenesis increase rate (BV/TV, 38.59 ± 7.75), almost 3 folds of 5-week samples (less than 2 folds for other groups). At 10 weeks, the BV/TV in the SFI-T/SFI-B group was about 3 folds of SFA-T/SFA-B groups (13.19 ± 2.59) and 4 folds of blank Control and SF/SF groups (13.65 ± 3.83 and 10.68 ± 2.57, respectively). According to the morphological analysis of bone trabeculae, Tb.N of SFA-T/SFA-B and SFI-T/SFI-B groups at 5 and 10 weeks demonstrated similar trends compared with BV/TV, while the Tb. Sp showed the reverse trends, which further confirmed the slowly co-released TGF-β3 and BMP-2 from the SFI-T/SFI-B scaffold potent promoted the repair of OC defects compared to surface-absorbed TGF-β3 and BMP-2.

Fig. 6
figure 6

The slowly co-released TGF-β3 and BMP-2 from SFI-T/SFI-B scaffolds continuously promoted the subchondral bone repair compared to surface-absorbed TGF-β3 and BMP-2. a–d 3D and 2D reconstructions based on Micro-CT data. Red circles and boxes indicated the borders of defect areas and the white imaging inside the circles and boxes represented the newly-formed bones. Scar bar = 5 mm. e, f Goldner stain. The asterisk in the Goldner stain (*) pictures indicated the neo-bone formation. g, h Immunohistochemistry stain of Runx2. The Brown stain in the pictures indicated the positive staining. i–k, m–o Quantitative analysis of relative bone volume (BV/TV), trabecular number (Tb. N), and trabecular Spacing (Tb. Sp) based on Micro-CT data (n = 4). l, p Quantitative analysis for Goldner stain (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Full size image
Histological and immunohistochemistry analyses

In the blank Control group, the defects at 5 weeks post-surgery were filled with fibroid tissue, and the surrounding tissue collapsed without the support of scaffolds. The defects of the other 3 groups were all covered with some cartilage-like tissue, validated by the histological and immunohistochemistry stains (Fig. 5b–d). Ten weeks post-surgery, a significantly stronger dense signal of GAG of the repaired cartilaginous layer was observed in SFI-T/SFI-B groups (about 4 folds of SFA-T/SFA-B groups). Moreover, the area of the Col-II positive region in the SFI-T/SFI-B groups was more significant than in the SFA-T/SFA-B groups. In both 5-week and 10-week samples, a few newly formed bones (orange-red stain in Fig. 6e, f) were indicated in Control, SF/SF, and SFA-T/SFA-B groups. Meanwhile, more neo-osseous tissues were stained in SFI-T/SFI-B groups, which were about 10 (5 weeks) and 20 (10 weeks) folds of SFA-T/SFA-B groups. The dense stain of Runx2 (Fig. 6g–h) in SFI-T/SFI-B groups further confirmed the higher osteogenic capacity of slowly co-released TGF-β3 and BMP-2 compared to that absorbed on the surface of SFA-T/SFA-B scaffolds.

The slowly co-release of TGF-β3 and BMP-2 from SFI-T/SFI-B scaffold enhanced the bioactivity of single-released TGF-β3 or BMP-2

To investigate the interaction effects between TGF-β3 and BMP-2, the OC defects in rabbits were treated (1) blank (Control group), (2) single-released TGF-β3 (SFI-T/SFI group), (3) single-released BMP-2 (SFI/SFI-B group), and (4) slowly co-released TGF-β3 and BMP-2 (SFI-T/SFI-B group). No visible adverse reactions were observed in the surgical area during the experiment periods. The samples were collected at 5 or 10 weeks post-surgery. Gross observation, micro-CT analyses and histological staining evaluations were used to assess the reconstruction of OC tissues.

Gross observation

As described above, the 5-week samples showed no regenerated tissues, and the border of the defect area collapsed in the blank Control group (Fig. 7a). The defect sites in the SFI-T/SFI-B group were filled with glossy white tissues, and few minor fractures could be observed. The defect site of the SFI-T/SFI groups is filled with opaque tissues, and a large hole can be observed in the center area. The photograph of the SFI/SFI-B group showed the defect zone was filled with undegraded materials and almost no neo-tissue was found when observed with the naked eyes. The 10-week samples from the SFI-T/SFI-B groups was repaired with smooth, well-integrated tissue and refined into hyaline cartilage-like tissue, which was not observed in the other groups. The SFI-T/SFI groups showed better repair of OC defects, filled with glossy tissues and left with few small cracks, compared to the SFI/SFI-B groups. The ICRS scores (Fig. 7e) of the blank Control group were 5.25 ± 1.32 and 2.88 ± 1.45, respectively for 5 weeks and 10 weeks. The scores of the SFI-T/SFI groups in both points (2.88 ± 1.32 for 5 weeks, 5.67 ± 1.44 for 10 weeks) were higher than the SFI/SFI-B groups (1.58 ± 1.04 for 5 weeks, 3.08 ± 0.92 for 10 weeks), but still significantly lower than SFI-T/SFI-B groups (6.20 ± 1.52 for 5 weeks, 10.00 ± 1.00 for 10 weeks). The above results demonstrated the potent chondrogenic bioactivity of TGF-β3 and the potential synergistic effects of BMP-2 on TGF-β3.

Fig. 7
figure 7

The slow co-release of TGF-β3 and BMP-2 from SFI-T/SFI-B scaffold enhanced the bioactivity of single-released TGF-β3. a Photographs of OC defects in the blank Control group and different scaffold groups. The red circle indicated the border of OC defect and inside was the regenerated tissue. b Histological H&E, c Safranin O & Fast Green, and d Col-II immunochemistry stain of OC defects. The red arrow in b indicated the border of the defect. The Brown stain in d indicated the positive stain. e Quantitative ICRS scoring analysis (n = 4). f Quantitative analysis for Safranin O & Fast Green stain (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Full size image
Micro-CT analyses

The reconstructed images of 5-week samples showed the defects of SFI-T/SFI groups were filled with tiny new subchondral bones, while much more glossy new subchondral tissues were observed in SFI/SFI-B and SFI-T/SFI-B groups (Fig. 8a–d). While the pictures from the 2D-x, y, z directions displayed the SFI/SFI-B group was mostly empty inside, and the neo-bone was less than SFI-T/SFI-B group showed in the 2D-x, y, z views, in which enhanced new subchondral bone density could be observed.

Fig. 8
figure 8

The slow co-release of TGF-β3 and BMP-2 from SFI-T/SFI-B scaffold enhanced the bioactivity of single-released BMP-2. a–d 3D and 2D reconstructions based on Micro-CT data. Red circles and boxes indicated the borders of defect areas and the white imaging inside the circles and boxes represented the newly-formed bones. Scar bar = 5 mm. e, f Goldner stain. The asterisk in the Goldner stain (*) pictures indicated the neo-bone formation. g–h Immunohistochemistry stain of Runx2. The Brown stain in the pictures indicated the positive staining. i–k, m–o Quantitative analysis of relative bone volume (BV/TV), trabecular number (Tb. N), and trabecular Spacing (Tb. Sp) based on Micro-CT data (n = 4). l, p Quantitative analysis for Goldner stain (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Full size image

Quantitatively, the blank control and SFI-T/SFI groups showed a similar bone mass (8.30 ± 1.11 and 7.77 ± 1.67, respectively) in 5-week samples (Fig. 8i–k, m–o), which was significantly lower than that of SFI/SFI-B and SFI-T/SFI-B groups (10.62 ± 0.96 and 12.4 ± 1.27, respectively). After 10 weeks, there was an increase in BV/TV in all groups. The BV/TV in the SFI-T/SFI-B group was about 1.5 folds of SFI/SFI-B groups (23.39 ± 3.49) and 2 folds of the SFI-T/SFI groups (17.6 ± 5.49). According to the morphological analysis of bone trabeculae, Tb. N of SFA-T/SFA-B and SFI-T/SFI-B groups at 5 weeks and 10 weeks demonstrated the similar trends compared with BV/TV, while the Tb. Sp showed the reverse trends, which further confirmed the powerful osteogenic capacity of BMP-2 and the possible synergistic effects of TGF-β3 on BMP-2.

Histological and immunohistochemistry analyses

The H&E and Safranin O & Fast Green stain revealed cartilage-like tissue in SFI-T/SFI-B and SFI-T/SFI groups, while fibrous-like tissue in control and SFI/SFI-B groups 5 weeks post-surgery (Fig. 7b–d). The expressions of GAG in control and SFI/SFI-B groups remained a lower level 10 weeks post-surgery. Both SFI-T/SFI-B and SFI-T/SFI groups at 10 weeks showed remarkably enhanced GAG signals compared to control groups, with fourfold and 2.7-fold increase, respectively. Furthermore, the SFI-T/SFI-B groups demonstrated more dense GAG stain than the SFI-T/SFI groups, which was consistent with the larger area and darker color of the Col-II positive region in SFI-T/SFI-B groups (Fig. 7c, d). Besides, some cartilage-like tissue in the SFI-T/SFI groups leaked to the subchondral bone area, disturbing the continuous boundary of the interface, which were not observed in SFI-T/SFI-B groups (Fig. 7b). In both 5-week samples and 10-week samples, little newly formed bones (orange red stain in Fig. 8e, f) were indicated in control and SFI-T/SFI groups. At 5 weeks, obvious neo-osseous tissues were stained in both SFI-T/SFI-B and SFI/SFI-B groups. At 10 weeks, SFI-T/SFI-B groups showed a larger stain area of newly formed bone than SFI/SFI-B groups. The dense stain of Runx2 in SFI-T/SFI-B groups further confirmed the higher osteogenic capacity of slowly co-released TGF-β3 and BMP-2 compared to that of single-released from of SFI/SFI-B groups scaffolds (Fig. 8g–h).

Discussions

The OC tissue made up of cartilage and subchondral bone, can transfer loads during weight bearing and joint motion. Any change in the composition or structure of this unit causes joint integrity to be disrupted and function to be lost [33, 34], which suggests the necessity and importance of regenerating cartilage and beneath subchondral bone simultaneously. Considering the regeneration of both cartilage and subchondral bone in OC defects necessitates diverse microenvironments, a bi-functional scaffold with chondrogenic and osteogenic bio-clues is needed [2, 35]. Herein, we fabricated BioCaP-coated SF scaffolds to sustainedly release TGF-β3 and BMP-2 to simulate the cartilage and bone formation microenvironment at the same time. The release kinetics of incorporated proteins from the scaffolds were determined as a function of time.

To be an ideal the scaffold applied in tissue engineering, SF should degrade gradually in the body to match the tissue growth rate. According to the USP, silk loses most of its tensile strength within 60 days post-implantation in vivo study. Within this definition, silk is correctly classified as a non-degradable biomaterial. However, in previous study published studies, the SF combined with nanosized BioCaP granules (SF-CaP complex) was degradable over 30 days in aqueous solution [36] and was degraded up to 22 ~ 43% in the enzymatic environment within 7 days [37]. The in vivo degradation was also observed [37]. After the 4-week subcutaneous implantation, the diameter of SF was reduced by 33.3%. When implanted in the bone defect sites, the diameter of SF was reduced by 25 ~ 66.7%. The above in vitro and in vivo results indicated the SF scaffolds can be considered as degradable biomaterials. The differences in the degradation profiles between USP and our previous studies could be attributed to the presence of BioCaP matrix. Calcium and phosphate ions dissolved could directly neutralize the protons. The more osteogenesis there was, the more the protons were generated and the more calcium and phosphate ions were needed to neutralize them. The osteogenic environment induced by BioCaP was more suitable for bone formation. Enhanced bone formation needed more calcium and phosphate ions, facilitating the degradation of SF-CaP complex [38].

The mechanical property of the scaffolds is one of the main issues when addressing bone and cartilage tissue regeneration. The inferior compression performance [22] limited the promotion of SF to regenerate OC defects. We combined the inorganic BioCaP coating to improve the mechanical properties of SF scaffolds. From the literature, the typical Young’s modulus for healthy cartilage was ranged from 500 to 17000 kPa [39]. Despite the different testing approach, the strength of the SF scaffold coated with BioCaP is comparable to that of human articular cartilage (Fig. 2e) conditions. However, the application of BioCaP coating did not change the cross-linking degree of SF according to our previous study [40], which may explain why there was no significant difference between these groups.

According to the literature, the cost-effective and stable Bovine serum albumin (BSA) is widely used as the model protein to determine the loading and release efficacy of different drug carrier system [41, 42]. According to the article published in Asian Journal of Pharmaceutical Sciences, 2021, BSA was adopted as the model protein in vitro to simulate the release of other protein, rhIFNα [43]. Our previously published studies also confirmed the BioCaP coating used in this study could fulfill the release of BSA. The loading and release efficacy of BSA were represented to demonstrate that of other proteins [30, 44,45,46,47]. Considering the TGF-β3 and BMP-2 are much more expensive and more difficult to be fabricated, we finally chose BSA as the model protein to simulate the loading rates of TGF-β3 and BMP-2 in order to decrease the bioactive waste and economic burden. The release rate was stable without remarkable burst release during the first 1 day, followed by a sustained release from day 7 to day 19. Only 66% of the protein incorporated into the scaffolds was slowly released during the 19-observation period. The stable and lasting release behavior of experimental scaffolds could match the continuous promotion of chondrogenesis and osteogenesis in following in vitro and in vivo experiments for SFI-T/SFI-B groups. The expressions of chondrogenesis (Col-II and SOX9) and osteogenesis (Col-I and OCN) relative genes at different intervals were always enhanced compared to other scaffolds. The enhancement was further confirmed in rabbit OC defect models. The SFI-T/SFI-B group with slowly co-released TGF-β3 and BMP-2 resulted in the best OC defect regeneration capacity when evaluated with ICRS scoring, micro-CT analysis, and Histological examinations.

Due to their broad clinical relevance, the profound delivery of TGF-β3 and BMP-2 was demonstrated as a proof of concept. Although their effects on the chondrogenesis and osteogenesis have been previously studied, sustained utilization of TGF-β3 and BMP-2 remains challenging due to their fast diffusion into the surrounding milieu and inactivation by enzymes. The BioCaP coated scaffold used in this study served as a slow-release delivery system. TGF-β3 and BMP-2 were incorporated into the BioCaP coated scaffolds during the coating deposition, thus protected by the BioCaP coatings. According to our previous studies, Xin Zhang et.al incorporated both BMP-2 and Icariin via the BioCaP coating system used in this study and tested whether the 2 proteins can be released together to exert their biological functions in a cranial bone defect model. The in vitro cell ALP activity assay, OCN expression assay and in vivo histomorphometric results confirmed that the incorporated 2 proteins can be both released to improve the bone tissue regeneration [23]. Based on their study, the TGF-β3 and BMP-2 were incorporated into the slow-release BioCaP coatings system separately. Then the two layers were stocked as upper and lower structure, which were glued together as one bilayer scaffold (SFI-T/SFI-B) using fibrin glue after fabrication. The bilayer scaffold was implant in the defect site and TGF-β3 and BMP-2 can be slowly co-released sustainably, which may be the reason for the best regeneration capacity in the SFI-T/SFI-B group. The SFA-T/SFA-TB group with burst release profile of TGF-β3 and BMP-2 showed no noticeable enhancement for Col-II, SOX9, Col-I, and OCN expressions compared to the control group since day 4. The newly formed bone (BV/TV) at 5 weeks in the SFA-T/SFA-B group was the smallest among all groups, possibly due to the abnormal osteoclast activation caused by the burst release of BMP-2 [48,49,50,51,52]. Another possible reason for the superior regeneration capacity in SFI-T/SFI-B group was the potential synergistic effects between TGF-β3 and BMP-2 compared to isolation application of TGF-β3 or BMP-2 in SFI-T/SFI and SFI/SFI-B groups. Shen et al. reported that the chondrogenic differentiation of BMSCs was reinforced by co-applicating TGF-β3 and BMP-2 compared with TGF-β3 alone [53]. Similarly, He et al. found increased OCN, OPG and OPN expression levels and ALP activity in BMSCs by overexpressing TGF-β3 and BMP-2 [54]. Except for in vitro studies, the improved recovery effect under TGF-β3 and BMP-2 was observed in the rat alveolar defect model [55]. These synergistic effects may be due to that the transcription of endogenous BMP-2 was enhanced in the presence of TGF-β3 [54, 56]. Meanwhile, applying BMP-2 may, in return, modulate the maturity of regenerated cartilage tissue [57]. The more specific mechanism underlying the synergistic effects between TGF-β3 and BMP-2 still needs more studies.

Calcium phosphate materials have been extensively employed to induce bone regeneration. The unique element composition and porous structure allow them to interact with signaling molecules and extracellular matrices when applied in vivo, creating an osteoconductive milieu [58, 59]. The presence of calcium phosphate in BioCaP@SF scaffolds (SFI-T/SFI-B, SFI/SFI-B and SFI-T/SFI) demonstrated more neo-bone formation (Micro-CT and Goldner stain analysis) and RUNX2 expression compared to control groups. However, some studies reported that calcium phosphate was reported to be associated with chondrocyte hypertrophic differentiation [60, 61]. It may influence the biological function of chondrocytes, consistent with the lowest macroscopic cartilage ICRS scores in SFI/SFI-B groups. To overcome the adverse effect of the chondrogenesis of calcium phosphate, Jung et al. modified the calcium phosphate-related biomaterials with chondrogenic biologic clues. They successfully resulted in cartilage regeneration [62, 63]. Inspired by the studies mentioned above, TGF-β3 was incorporated in the SFI-T/SFI and SFI-T/SFI-B group in this study to alleviate or even reverse the unfavorable effects. The results showed that the SFI-T/SFI and SFI-T/SFI-B groups made up the limitations of calcium phosphates and led to superior chondrogenesis, as indicated in the Safranin O & Fast Green stain.

Besides, this study displayed some limitations. Firstly, a more long-term in vivo study is needed to further confirm the long-lasting effects and final repair effects of SFI-T/SFI-B scaffolds and a shame group is still needed to represent as the positive control to reflect the extent of regeneration capacity of implanted scaffold. Secondly, the protein loading rate and slow-release profile in this study were checked with BSA, instead of TGF-β3 and BMP-2. Direct check of loading rate and slow-release profile for TGF-β3 and BMP-2 will be required further. Lastly, further studies will be carried out in the future to confirm the signaling pathways involved further.

Conclusions

In this study, we prepared a TGF-β3 and BMP-2 slowly co-released bi-functional scaffolds for osteochondral tissue regeneration using a BioCaP coating system. The biological effects of burst and slow release were tested with surface absorption mode and BioCaP coating incorporated mode, respectively. The slow release from SFI-T/SFI-B scaffolds maintained better chondrogenesis and osteogenesis than burst release in vitro. Moreover, the slowly co-released TGF-β3 and BMP-2 from SFI-T/SFI-B scaffolds obtained more cartilage and neo-bone formation compared to burst release or single-release mode. This approach may contribute to the clinical translation of controlled drug delivery in tissue engineering in the future.

Availability of data and materials

We declare that the data of this article are original and will be fully available and obtained from the corresponding author.

Change history

References

  1. Logerstedt DS, Scalzitti DA, Bennell KL, Hinman RS, Silvers-Granelli H, Ebert J, Hambly K, Carey JL, Snyder-Mackler L, Axe MJ, et al. Knee pain and mobility impairments: meniscal and articular cartilage lesions revision 2018. J Orthop Sports Phys Ther. 2018;48(2):A1–50. https://doi.org/10.2519/jospt.2018.0301.

    Article  PubMed  Google Scholar 

  2. Doyle SE, Snow F, Duchi S, O’Connell CD, Onofrillo C, Di Bella C, Pirogova E. 3D printed multiphasic scaffolds for osteochondral repair: challenges and opportunities. Int J Mol Sci. 2021. https://doi.org/10.3390/ijms222212420.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Ondresik M, Oliveira JM, Reis RL. Advances for treatment of knee OC defects. Adv Exp Med Biol. 2018;1059:3–24. https://doi.org/10.1007/978-3-319-76735-2_1.

    Article  CAS  PubMed  Google Scholar 

  4. Xiao H, Huang W, Xiong K, Ruan S, Yuan C, Mo G, Tian R, Zhou S, She R, Ye P, et al. Osteochondral repair using scaffolds with gradient pore sizes constructed with silk fibroin, chitosan, and nano-hydroxyapatite. Int J Nanomedicine. 2019;14:2011–27. https://doi.org/10.2147/IJN.S191627.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhang W, Ling C, Zhang A, Liu H, Jiang Y, Li X, Sheng R, Yao Q, Chen J. An all-silk-derived functional nanosphere matrix for sequential biomolecule delivery and in situ osteochondral regeneration. Bioact Mater. 2020;5(4):832–43. https://doi.org/10.1016/j.bioactmat.2020.05.003.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Kiernan CH, Wolvius EB, Brama PAJ, Farrell E. The immune response to allogeneic differentiated mesenchymal stem cells in the context of bone tissue engineering. Tissue Eng Part B Rev. 2018;24(1):75–83. https://doi.org/10.1089/ten.TEB.2017.0175.

    Article  PubMed  Google Scholar 

  7. Zhai M, Zhu Y, Yang M, Mao C. Human mesenchymal stem cell derived exosomes enhance cell-free bone regeneration by altering their miRNAs profiles. Adv Sci. 2020;7(19):2001334. https://doi.org/10.1002/advs.202001334.

    Article  CAS  Google Scholar 

  8. Zhao T, Zhang ZN, Rong Z, Xu Y. Immunogenicity of induced pluripotent stem cells. Nature. 2011;474(7350):212–5. https://doi.org/10.1038/nature10135.

    Article  CAS  PubMed  Google Scholar 

  9. Cernigliaro V, Peluso R, Zedda B, Silengo L, Tolosano E, Pellicano R, Altruda F, Fagoonee S. Evolving cell-based and cell-free clinical strategies for treating severe human liver diseases. Cells. 2020. https://doi.org/10.3390/cells9020386.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Bozhokin MS, Sopova YV, Kachkin DV, Rubel AA, Khotin MG. Mechanisms of TGFbeta3 action as a therapeutic agent for promoting the synthesis of extracellular matrix proteins in hyaline cartilage. Biochemistry. 2020;85(4):436–47. https://doi.org/10.1134/S0006297920040045.

    Article  CAS  PubMed  Google Scholar 

  11. Patil AS, Sable RB, Kothari RM. An update on transforming growth factor-beta (TGF-beta): sources, types, functions and clinical applicability for cartilage/bone healing. J Cell Physiol. 2011;226(12):3094–103. https://doi.org/10.1002/jcp.22698.

    Article  CAS  PubMed  Google Scholar 

  12. Deng M, Mei T, Hou T, Luo K, Luo F, Yang A, Yu B, Pang H, Dong S, Xu J. TGFbeta3 recruits endogenous mesenchymal stem cells to initiate bone regeneration. Stem Cell Res Ther. 2017;8(1):258. https://doi.org/10.1186/s13287-017-0693-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sahu N, Baligar P, Midha S, Kundu B, Bhattacharjee M, Mukherjee S, Mukherjee S, Maushart F, Das S, Loparic M, et al. Nonmulberry silk fibroin scaffold shows superior osteoconductivity than mulberry silk fibroin in calvarial bone regeneration. Adv Healthcare Mater. 2015;4(11):1709–21. https://doi.org/10.1002/adhm.201500283.

    Article  CAS  Google Scholar 

  14. Lee CH, Cook JL, Mendelson A, Moioli EK, Yao H, Mao JJ. Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study. Lancet. 2010;376(9739):440–8. https://doi.org/10.1016/s0140-6736(10)60668-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yang Z, Li H, Tian Y, Fu L, Gao C, Zhao T, Cao F, Liao Z, Yuan Z, Liu S, et al. Biofunctionalized structure and ingredient mimicking scaffolds achieving recruitment and chondrogenesis for staged cartilage regeneration. Front Cell Dev Biol. 2021;9:655440. https://doi.org/10.3389/fcell.2021.655440.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol. 2016;12(4):203–21. https://doi.org/10.1038/nrendo.2016.12.

    Article  CAS  PubMed  Google Scholar 

  17. Florczak A, Deptuch T, Kucharczyk K, Dams-Kozlowska H. Systemic and local silk-based drug delivery systems for cancer therapy. Cancers. 2021. https://doi.org/10.3390/cancers13215389.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Khosropanah MH, Vaghasloo MA, Shakibaei M, Mueller AL, Kajbafzadeh AM, Amani L, Haririan I, Azimzadeh A, Hassannejad Z, Zolbin MM. Biomedical applications of silkworm (Bombyx Mori) proteins in regenerative medicine (a narrative review). J Tissue Eng Regen Med. 2021. https://doi.org/10.1002/term.3267.

    Article  PubMed  Google Scholar 

  19. Kim DW, Hwang HS, Kim DS, Sheen SH, Heo DH, Hwang G, Kang SH, Kweon H, Jo YY, Kang SW, et al. Effect of silk fibroin peptide derived from silkworm Bombyx mori on the anti-inflammatory effect of Tat-SOD in a mice edema model. BMB Rep. 2011;44(12):787–92. https://doi.org/10.5483/bmbrep.2011.44.12.787.

    Article  CAS  PubMed  Google Scholar 

  20. Gou S, Huang Y, Sung J, Xiao B, Merlin D. Silk fibroin-based nanotherapeutics: application in the treatment of colonic diseases. Nanomedicine. 2019;14(17):2373–8. https://doi.org/10.2217/nnm-2019-0058.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lee J, Byun H, Madhurakkat Perikamana SK, Lee S, Shin H. Current advances in immunomodulatory biomaterials for bone regeneration. Adv Healthc Mater. 2019;8(4):e1801106. https://doi.org/10.1002/adhm.201801106.

    Article  CAS  PubMed  Google Scholar 

  22. Zhao Y, Zhu ZS, Guan J, Wu SJ. Processing, mechanical properties and bio-applications of silk fibroin-based high-strength hydrogels. Acta Biomater. 2021;125:57–71. https://doi.org/10.1016/j.actbio.2021.02.018.

    Article  CAS  PubMed  Google Scholar 

  23. Zhang X, Lin X, Liu T, Deng L, Huang Y, Liu Y. Osteogenic enhancement between icariin and bone morphogenetic protein 2: a potential osteogenic compound for bone tissue engineering. Front Pharmacol. 2019;10:201. https://doi.org/10.3389/fphar.2019.00201.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Teng F, Wei L, Yu D, Deng L, Zheng Y, Lin H, Liu Y. Vertical bone augmentation with simultaneous implantation using deproteinized bovine bone block functionalized with a slow delivery of BMP-2. Clin Oral Implants Res. 2020;31(3):215–28. https://doi.org/10.1111/clr.13558.

    Article  PubMed  Google Scholar 

  25. Kokubo T, Ito S, Huang ZT, Hayashi T, Sakka S, Kitsugi T, Yamamuro T. Ca, P-rich layer formed on high-strength bioactive glass-ceramic A-W. J Biomed Mater Res. 1990;24(3):331–43. https://doi.org/10.1002/jbm.820240306.

    Article  CAS  PubMed  Google Scholar 

  26. Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W. J Biomed Mater Res. 1990;24(6):721–34. https://doi.org/10.1002/jbm.820240607.

    Article  CAS  PubMed  Google Scholar 

  27. Lin X, Chen J, Liao Y, Pathak JL, Li H, Liu Y. Biomimetic calcium phosphate coating as a drug delivery vehicle for bone tissue engineering: a mini-review. Coatings. 2020;10(11):1118.

    Article  CAS  Google Scholar 

  28. Kundu B, Brancato V, Oliveira JM, Correlo VM, Reis RL, Kundu SC. Silk fibroin promotes mineralization of gellan gum hydrogels. Int J Biol Macromol. 2020;153:1328–34. https://doi.org/10.1016/j.ijbiomac.2019.10.269.

    Article  CAS  PubMed  Google Scholar 

  29. Wu G, Liu Y, Iizuka T, Hunziker EB. The effect of a slow mode of BMP-2 delivery on the inflammatory response provoked by bone-defect-filling polymeric scaffolds. Biomaterials. 2010;31(29):7485–93. https://doi.org/10.1016/j.biomaterials.2010.06.037.

    Article  CAS  PubMed  Google Scholar 

  30. Wernike E, Hofstetter W, Liu Y, Wu G, Sebald HJ, Wismeijer D, Hunziker EB, Siebenrock KA, Klenke FM. Long-term cell-mediated protein release from calcium phosphate ceramics. J Biomed Mater Res A. 2010;92(2):463–74. https://doi.org/10.1002/jbm.a.32411.

    Article  CAS  PubMed  Google Scholar 

  31. Liu T, Wu G, Zheng Y, Wismeijer D, Everts V, Liu Y. Cell-mediated BMP-2 release from a novel dual-drug delivery system promotes bone formation. Clin Oral Implants Res. 2014;25(12):1412–21. https://doi.org/10.1111/clr.12283.

    Article  PubMed  Google Scholar 

  32. Saha S, Kundu B, Kirkham J, Wood D, Kundu SC, Yang XB. Osteochondral tissue engineering in vivo: a comparative study using layered silk fibroin scaffolds from mulberry and nonmulberry silkworms. PLoS ONE. 2013;8(11):e80004. https://doi.org/10.1371/journal.pone.0080004.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Goldring SR, Goldring MB. Changes in the osteochondral unit during osteoarthritis: structure, function and cartilage-bone crosstalk. Nat Rev Rheumatol. 2016;12(11):632–44. https://doi.org/10.1038/nrrheum.2016.148.

    Article  PubMed  Google Scholar 

  34. Liao J, Tian T, Shi S, Xie X, Ma Q, Li G, Lin Y. The fabrication of biomimetic biphasic CAN-PAC hydrogel with a seamless interfacial layer applied in osteochondral defect repair. Bone Res. 2017;5:17018. https://doi.org/10.1038/boneres.2017.18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kang H, Zeng Y, Varghese S. Functionally graded multilayer scaffolds for in vivo osteochondral tissue engineering. Acta Biomater. 2018;78:365–77. https://doi.org/10.1016/j.actbio.2018.07.039.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yan LP, Oliveira JM, Oliveira AL, Caridade SG, Mano JF, Reis RL. Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications. Acta Biomater. 2012;8(1):289–301. https://doi.org/10.1016/j.actbio.2011.09.037.

    Article  CAS  PubMed  Google Scholar 

  37. Yan LP, Silva-Correia J, Oliveira MB, Vilela C, Pereira H, Sousa RA, Mano JF, Oliveira AL, Oliveira JM, Reis RL. Bilayered silk/silk-nanoCaP scaffolds for osteochondral tissue engineering: in vitro and in vivo assessment of biological performance. Acta Biomater. 2015;12:227–41. https://doi.org/10.1016/j.actbio.2014.10.021.

    Article  CAS  PubMed  Google Scholar 

  38. Wang D, Tabassum A, Wu G, Deng L, Wismeijer D, Liu Y. Bone regeneration in critical-sized bone defect enhanced by introducing osteoinductivity to biphasic calcium phosphate granules. Clin Oral Implants Res. 2017;28(3):251–60. https://doi.org/10.1111/clr.12791.

    Article  CAS  PubMed  Google Scholar 

  39. Means AK, Shrode CS, Whitney LV, Ehrhardt DA, Grunlan MA. Double network hydrogels that mimic the modulus, strength, and lubricity of cartilage. Biomacromol. 2019;20(5):2034–42. https://doi.org/10.1021/acs.biomac.9b00237.

    Article  CAS  Google Scholar 

  40. Yan LP, Silva-Correia J, Correia C, Caridade SG, Fernandes EM, Sousa RA, Mano JF, Oliveira JM, Oliveira AL, Reis RL. Bioactive macro/micro porous silk fibroin/nano-sized calcium phosphate scaffolds with potential for bone-tissue-engineering applications. Nanomedicine. 2013;8(3):359–78. https://doi.org/10.2217/nnm.12.118.

    Article  CAS  PubMed  Google Scholar 

  41. Determan AS, Trewyn BG, Lin VS, Nilsen-Hamilton M, Narasimhan B. Encapsulation, stabilization, and release of BSA-FITC from polyanhydride microspheres. J Control Release. 2004;100(1):97–109. https://doi.org/10.1016/j.jconrel.2004.08.006.

    Article  CAS  PubMed  Google Scholar 

  42. Zhu Y, Yao L, Liu Z, Weng W, Cheng K. Electrical potential specified release of BSA/Hep/polypyrrole composite film and its cellular responses. ACS Appl Mater Interfaces. 2019;11(28):25457–64. https://doi.org/10.1021/acsami.9b09333.

    Article  CAS  PubMed  Google Scholar 

  43. Zhou Z, Zhang S, Yang G, Gao Y. Enhanced delivery efficiency and sustained release of biopharmaceuticals by complexation-based gel encapsulated coated microneedles: rhIFNα-1b example. Asian J Pharm Sci. 2021;16(5):612–22. https://doi.org/10.1016/j.ajps.2021.05.002.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Wernike E, Montjovent MO, Liu Y, Wismeijer D, Hunziker EB, Siebenrock KA, Hofstetter W, Klenke FM. VEGF incorporated into calcium phosphate ceramics promotes vascularisation and bone formation in vivo. Eur Cell Mater. 2010;19:30–40. https://doi.org/10.2220/ecm.v019a04.

    Article  CAS  PubMed  Google Scholar 

  45. Zheng Y, Wu G, Liu T, Liu Y, Wismeijer D, Liu Y. A novel BMP2-coprecipitated, layer-by-layer assembled biomimetic calcium phosphate particle: a biodegradable and highly efficient osteoinducer. Clin Implant Dent Relat Res. 2014;16(5):643–54. https://doi.org/10.1111/cid.12050.

    Article  PubMed  Google Scholar 

  46. Liu Y, Schouten C, Boerman O, Wu G, Jansen JA, Hunziker EB. The kinetics and mechanism of bone morphogenetic protein 2 release from calcium phosphate-based implant-coatings. J Biomed Mater Res A. 2018;106(9):2363–71. https://doi.org/10.1002/jbm.a.36398.

    Article  CAS  PubMed  Google Scholar 

  47. Wei L, Yu D, Wang M, Deng L, Wu G, Liu Y. Dose effects of slow-released bone morphogenetic protein-2 functionalized β-tricalcium phosphate in repairing critical-sized bone defects. Tissue Eng Part A. 2020;26(3–4):120–9. https://doi.org/10.1089/ten.TEA.2019.0161.

    Article  CAS  PubMed  Google Scholar 

  48. Chen C, Huang K, Zhu J, Bi Y, Wang L, Jiang J, Zhu T, Yan X, Zhao J. A novel elastic and controlled-release poly(ether-ester-urethane)urea scaffold for cartilage regeneration. J Mater Chem B. 2020;8(18):4106–21. https://doi.org/10.1039/c9tb02754h.

    Article  CAS  PubMed  Google Scholar 

  49. Xu Y, Yang Y, Hua Z, Li S, Yang Z, Liu Q, Fu G, Ji P, Wu Q. BMP2 immune complexes promote new bone formation by facilitating the direct contact between osteoclasts and osteoblasts. Biomaterials. 2021;275:120890. https://doi.org/10.1016/j.biomaterials.2021.120890.

    Article  CAS  PubMed  Google Scholar 

  50. Cho TH, Kim IS, Lee B, Park SN, Ko JH, Hwang SJ. (*) Early and marked enhancement of new bone quality by alendronate-loaded collagen sponge combined with bone morphogenetic protein-2 at high dose: a long-term study in calvarial defects in a rat model. Tissue Eng Part A. 2017;23(23–24):1343–60. https://doi.org/10.1089/ten.TEA.2016.0557.

    Article  CAS  PubMed  Google Scholar 

  51. Hu T, Naidu M, Yang Z, Lam WM, Kumarsing RA, Ren X, Ng F, Wang M, Liu L, Tan KC, et al. Bone regeneration by controlled release of bone morphogenetic protein-2: a rabbit spinal fusion chamber molecular study. Tissue Eng Part A. 2019;25(19–20):1356–68. https://doi.org/10.1089/ten.TEA.2018.0281.

    Article  CAS  PubMed  Google Scholar 

  52. Jarrar H, Çetin Altındal D, Gümüşderelioğlu M. Effect of melatonin/BMP-2 co-delivery scaffolds on the osteoclast activity. J Mater Sci Mater Med. 2021;32(4):32. https://doi.org/10.1007/s10856-021-06502-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Shen B, Wei A, Tao H, Diwan AD, Ma DDF. BMP-2 enhances TGF-β3–mediated chondrogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in alginate bead culture. Tissue Eng Part A. 2008;15(6):1311–20. https://doi.org/10.1089/ten.tea.2008.0132.

    Article  Google Scholar 

  54. He W, Chen L, Huang Y, Xu Z, Xu W, Ding N, Chen J. Synergistic effects of recombinant lentiviral-mediated BMP2 and TGF-beta3 on the osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro. Cytokine. 2019;120:1–8. https://doi.org/10.1016/j.cyto.2019.03.020.

    Article  CAS  PubMed  Google Scholar 

  55. Alici-Garipcan A, Korkusuz P, Bilgic E, Askin K, Aydin HM, Ozturk E, Inci I, Ozkizilcik A, Kamile Ozturk K, Piskin E, et al. Critical-size alveolar defect treatment via TGF-ß3 and BMP-2 releasing hybrid constructs. J Biomater Sci Polym Ed. 2019;30(5):415–36. https://doi.org/10.1080/09205063.2019.1571397.

    Article  CAS  PubMed  Google Scholar 

  56. Klar RM, Duarte R, Dix-Peek T, Ripamonti U. The induction of bone formation by the recombinant human transforming growth factor-β3. Biomaterials. 2014;35(9):2773–88. https://doi.org/10.1016/j.biomaterials.2013.12.062.

    Article  CAS  PubMed  Google Scholar 

  57. Gamer LW, Pregizer S, Gamer J, Feigenson M, Ionescu A, Li Q, Han L, Rosen V. The role of Bmp2 in the maturation and maintenance of the murine knee joint. J Bone Miner Res. 2018;33(9):1708–17. https://doi.org/10.1002/jbmr.3441.

    Article  CAS  PubMed  Google Scholar 

  58. Tang Z, Li X, Tan Y, Fan H, Zhang X. The material and biological characteristics of osteoinductive calcium phosphate ceramics. Regen Biomater. 2018;5(1):43–59. https://doi.org/10.1093/rb/rbx024.

    Article  CAS  PubMed  Google Scholar 

  59. Humbert P, Brennan MA, Davison N, Rosset P, Trichet V, Blanchard F, Layrolle P. Immune modulation by transplanted calcium phosphate biomaterials and human mesenchymal stromal cells in bone regeneration. Front Immunol. 2019;10:663. https://doi.org/10.3389/fimmu.2019.00663.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Meyer F, Dittmann A, Kornak U, Herbster M, Pap T, Lohmann CH, Bertrand J. Chondrocytes from osteoarthritic and chondrocalcinosis cartilage represent different phenotypes. Front Cell Dev Biol. 2021;9:622287. https://doi.org/10.3389/fcell.2021.622287.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Stücker S, Bollmann M, Garbers C, Bertrand J. The role of calcium crystals and their effect on osteoarthritis pathogenesis. Best Pract Res Clin Rheumatol. 2021;35(4):101722. https://doi.org/10.1016/j.berh.2021.101722.

    Article  PubMed  Google Scholar 

  62. Jung A, Makkar P, Amirian J, Lee BT. A novel hybrid multichannel biphasic calcium phosphate granule-based composite scaffold for cartilage tissue regeneration. J Biomater Appl. 2018;32(6):775–87. https://doi.org/10.1177/0885328217741757.

    Article  CAS  PubMed  Google Scholar 

  63. Kumai T, Yui N, Yatabe K, Sasaki C, Fujii R, Takenaga M, Fujiya H, Niki H, Yudoh K. A novel, self-assembled artificial cartilage-hydroxyapatite conjugate for combined articular cartilage and subchondral bone repair: histopathological analysis of cartilage tissue engineering in rat knee joints. Int J Nanomed. 2019;14:1283–98. https://doi.org/10.2147/ijn.S193963.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Prof. Xuebin Yang from the School of Dentistry, University of Leeds, for promoting collaborations between Nanjing Stomatological Hospital and 3Bs Research Group, I3Bs -University of Minho.

Funding

This work was supported by the National Natural Science Foundation of China [81670960], Jiangsu Provincial Medical Youth Talent [QNRC2016115], Natural Science Foundation of Jiangsu Province [SBK2021021787], and Top Young and Middle-aged Innovative Talents Program of Nanjing [ZKX20048].

Author information

Authors and Affiliations

  1. Orthodontic Department, Nanjing Stomatological Hospital, Affiliated hospital of Medical School, Institute of Stomatology, Nanjing University, No. 30 Zhongyang Road, Nanjing, Jiangsu, China

    Jiping Chen, Yanyi Wang, Baochao Li & Huang Li

  2. Department of Stomatology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, No. 321 Zhongshan Road, Nanjing, Jiangsu, China

    Jiping Chen

  3. Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, Royal Free Hospital, Rowland Hill Street, London, NW3 2PF, UK

    Tianyi Tang

  4. 3B’s Research Group, I3Bs-Research Institute On Biomaterials, Biodegradables and Biomimetics of University of Minho, Headquarters of the European Institute of Excellence On Tissue Engineering and Regenerative Medicine, Avepark, Parque de Ciência E Tecnologia, Zona Industrial da Gandra, Barco, 4805-017, Guimarães, Portugal

    Banani Kundu, Subhas C. Kundu & Rui L. Reis

  5. ICVS/3B’s - PT Government Associate Laboratory, Braga, Guimarães, Portugal

    Banani Kundu, Subhas C. Kundu & Rui L. Reis

  6. Department of Biotechnology, Adamas University, Kolkata, 700126, India

    Banani Kundu

  7. School/Hospital of Stomatology, Zhejiang Chinese Medical University, No.548 Binwen Road, Hangzhou, 310053, China

    Xingnan Lin

Authors
  1. Jiping Chen
    View author publications

    Search author on:PubMed Google Scholar

  2. Yanyi Wang
    View author publications

    Search author on:PubMed Google Scholar

  3. Tianyi Tang
    View author publications

    Search author on:PubMed Google Scholar

  4. Baochao Li
    View author publications

    Search author on:PubMed Google Scholar

  5. Banani Kundu
    View author publications

    Search author on:PubMed Google Scholar

  6. Subhas C. Kundu
    View author publications

    Search author on:PubMed Google Scholar

  7. Rui L. Reis
    View author publications

    Search author on:PubMed Google Scholar

  8. Xingnan Lin
    View author publications

    Search author on:PubMed Google Scholar

  9. Huang Li
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Conceptualisation: Huang Li. Silk protein fibroin fabrication: Rui L. Reis, Banani Kundu and Subhas C. Kundu. BioCaP coating deposition: Jiping Chen and Xingnan Lin. In vitro experiments: Jiping Chen and Yanyi Wang. In vivo rabbit experiments: Jiping Chen, Yanyi Wang, Tianyi Tang and Baochao Li. Data analyses: Jiping Chen and Yanyi Wang. Formal analysis: Jiping Chen. Supervision: Huang Li. Draft writing: Jiping Chen and Huang Li. Language modification: Banani Kundu and Subhas C. Kundu. Draft review: all authors.

Corresponding authors

Correspondence to Xingnan Lin or Huang Li.

Ethics declarations

Ethics approval and consent to participate

The Institutional Ethics Committee of Nanjing Agricultural University approved the animal study with the registration number PT2021003. All animal study procedures were performed according to the ethical rules and regulations of Nanjing Agriculture University.

Competing interests

The authors declare there is no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The original online version of this article was revised: Authors affiliations and the affiliation assignment to the authors has also changed.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, J., Wang, Y., Tang, T. et al. Enhanced effects of slowly co-released TGF-β3 and BMP-2 from biomimetic calcium phosphate-coated silk fibroin scaffolds in the repair of osteochondral defects. J Nanobiotechnol 22, 453 (2024). https://doi.org/10.1186/s12951-024-02712-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12951-024-02712-0

Keywords

Journal of Nanobiotechnology

ISSN: 1477-3155

Contact us