Introduction

Articular cartilage (AC) plays a crucial role in maintaining joint motion. However, due to its limited regeneration capacity, AC damage caused by sports injury, trauma and ageing may eventually lead to osteoarthritis (OA) [1, 2]. Currently, the long-term prognostic effects of common therapeutic strategies, including arthroscopic debridement, microfracture (MF), or autologous chondrocyte implantation (ACI), are still unsatisfactory [3]. In recent years, research on in situ AC tissue engineering techniques with endogenous stem cells has made good progress [4]. The main factors affecting AC in situ repair include the destruction of the regenerative microenvironment in the defect area and an insufficient number of stem cells [5, 6]. In situ tissue engineering uses biological materials or bioactive factors to improve the regeneration microenvironment and recruit more endogenous stem cells from the joint cavity to the defect area to promote cartilage regeneration [7].

Among many biomaterials, decellularized cartilage extracellular matrix (ECM) has been widely used because of its outstanding biocompatibility and biochemical cue retention [8, 9]. Previous studies by our group have also demonstrated the positive effect of ECM on cartilage regeneration [10, 11]. However, traditional ECM scaffolds are usually lyophilized. In addition to their poor mechanical properties, their uneven pore size affects further ECM application. As a representative technology for precise scaffold fabrication, 3D printing is widely used in the construction of cartilage tissue engineering scaffolds [12, 13]. However, traditional high-temperature-melt fused deposition modelling (FDM) printing technology usually destroys the chemical structure of the material, leading to a capacity reduction [14]. Low-temperature deposition manufacturing (LDM) provides a possibility for scaffold fabrication while retaining the biological activity of the materials [15]. Unlike in FDM, bioinks are ejected from the printing nozzle and immediately solidify on a cryogenic platform [16]. After lyophilization, the bioinks form a hierarchical porous structure [17]. The interconnected porous structure and high porosity of scaffolds are not only essential for cell adhesion and growth but are also beneficial for oxygen and nutrient diffusion [15, 17]. In particular, recent studies have shown that scaffolds supported with macropores and micro/nanostructures perform better than only macroporous scaffolds in vivo [17, 18]. This hierarchical structure provides more adsorption sites for bioactive molecules and improves nutrient and metabolic waste transport. Chen et al. mixed ECM and waterborne polyurethane (WPU) to make bioinks for cryogenic 3D printing, constructed a hybrid scaffold and showed excellent repair effects in vivo [14]. However, the addition of synthetic materials still destroys the biochemical cues of the ECM itself, and making pure ECM bioink and printing it into scaffolds using LDM seems to better preserve its active properties. In addition, to show the advantage of the new scaffold (LDM) over the traditional scaffold (FDM), we added the FDM structure group to the experimental groups compared in the Supplementary File.

An ideal cartilage tissue-engineered scaffold must be biocompatible and porous while also having sufficient biological stimulation [19, 20]. Therefore, to improve overall scaffold performance, we need to supplement the corresponding bioactive factors. GDF-5, a member of the TGF-β/BMP superfamily, is essential for prechondrogenic mesenchymal stem cell (MSC) cohesion and directly affects cartilage and bone development [21, 22]. Recently, some studies have shown that GDF-5 protein significantly promotes bone marrow mesenchymal stem cell (BMSC) migration and chondrogenic differentiation [23, 24]. Therefore, GDF-5 was introduced to improve the scaffold recruitment of MSCs and chondrogenic differentiation.

As shown in Fig. 1, we first prepared a pure ECM bioink with an optimal concentration, which was then mixed with GDF-5 and coprinted by LDM to fabricate a hierarchical porous scaffold. The functional scaffold not only provided the necessary microenvironment for articular cartilage regeneration but also promoted more MSC migration to the defect area and further enhanced chondrogenic differentiation through sustained release of GDF-5. Furthermore, we also evaluated the regenerative potential of the hierarchical porous ECM/GDF-5 scaffold in vivo and discussed its prospects for future application. To the best of our knowledge, this is the first report on the construction of cartilage tissue engineering scaffolds by LDM using pure ECM as the main bioink component, and we believe that our findings will provide a new treatment strategy for AC defect repair.

Fig. 1
figure 1

Schematic illustration of the study design

Full size image

Materials and methods

Preparation and characterization of ECM bioinks

Preparation of ECM bioinks

Decellularized cartilage ECM was prepared by a combination of chemical and physical methods based on previous studies with slight modifications [10, 11, 25]. The specific experimental procedure is detailed in Supplementary File section 1.1. The ECM homogenate was lyophilized and then broken and ground into dry powder, and then 3, 5, 7, 9, and 11% (mass/volume) ECM was selected for preliminary bioink exploration. Specifically, 3.0 g, 5.0 g, 7.0 g, 9.0 g, and 11.0 g of dried cartilage ECM was weighed and added to beakers containing 100 ml of 5% acetic acid solution. Next, ECM bioinks at different concentrations were obtained by crushing the material in an ice bath at 75% amplitude for a total of 2 min using a Q125 ultrasonograph (Qsonica, USA).

Gelation status and particle size of ECM bioinks

The gelation status of ECM bioinks at different concentrations was assessed by a dispensing needle to examine the printability.

To further evaluate the printability of ECM bioinks, we examined their particle size using dynamic light scattering (Delsa™ Nano C Particle Analyser).

Fourier transform infrared (FTIR) spectroscopy

The functional groups of the lyophilized ECM bioinks at different concentrations were identified using a Bruker Tensor 27 FTIR spectrometer (Nicolet IS5, Germany). The samples were tested in reflection mode. All spectra were recorded between 4000 and 500 cm − 1 with a resolution of 1 cm − 1.

Rheological characterization of ECM bioinks

As a crucial indicator for assessing the printability of bioinks, the rheological properties of ECM bioinks were tested with a Paar MCR 301 advanced rotational rheometer (Austria) with a diameter of 20 mm. The storage and loss moduli of bioinks were tested at 25 °C at 101-103 Hz. The shear viscosity of the bioinks was measured at different concentrations at an angular velocity of 101-102.

Preparation and characterization of pure ECM scaffolds

Preparation of pure ECM scaffolds

We prepared pure ECM scaffolds with different ECM concentrations by an LDM 3D printing system. The abovementioned ECM bioinks at concentrations of 5, 7, and 9%, which were suitable for printing, were loaded into a 3D printer fitted with a plastic syringe with ink and equipped with a low-temperature receiver plate. Briefly, the printer followed a predesigned printing route for the scaffold model, and the bioink was layered through the printhead and deposited on a cryogenic receiver plate at − 30 °C. Then, the extruded fibre diameter was quickly cured during the printing process. All printed scaffolds were transferred to − 80 °C and lyophilized in a freeze dryer for 48 hours to remove residual solvents. Finally, all scaffolds were sterilized by cobalt 60 and used for subsequent biological experiments.

Scanning Electron Microscopy (SEM) and particle size distribution

To evaluate the microstructures of the ECM scaffolds with different ECM concentrations, we observed them by scanning electron microscopy (SEM, S-4800, Hitachi, Japan). In brief, the freeze-dried ECM scaffolds were fixed and sprayed with gold. The microstructures were observed and photographed under an electron microscope, and the micropore size distributions of the scaffolds were further analysed by ImageJ software.

Porosity measurement and biomechanical assay

To measure the porosity of the scaffolds, the classical ethanol replacement method was used in this study. Detailed experimental procedures are available in Supplementary File section 1.2.

To evaluate the biomechanical properties of the ECM scaffolds with different ECM concentrations, 5*5*1.2 mm3 scaffolds were prepared, with 3 scaffolds per concentration. The mechanical strength of the scaffold was tested using a BOSE biomechanical testing machine (BOSE 5100, USA). We assessed the compressive strength of the scaffolds by plotting a strain–stress curve, and the compression modulus was defined according to the linear slope of the strain–stress curve.

In vitro cytocompatibility studies of pure ECM scaffolds

Isolation, culture and identification of BMSCs

This study was approved by the Animal Ethics Committee of the General Hospital of the Chinese People’s Liberation Army. BMSC isolation and culture were performed according to a previously described method [15, 16].

In our study, for the first time, a cartilage scaffold with a graded porous structure was prepared by using pure ECM as a bioink in LDM 3D printing. Through experiments, we found that with the increase in ECM concentration in the solvent, the bioink gradually became overgelated, and the most suitable bioinks for printing had 5, 7 and 9% ECM. After the scaffolds were printed by using 5, 7 and 9% ECM bioinks, we found that all the scaffolds had hierarchical porous structures, which could not only promote the exchange of nutrients and oxygen but also provide attachment and adsorption for MSCs and bioactive molecules. In addition, the pore sizes of the three scaffolds decreased gradually with increasing ECM concentration. Previous studies have revealed that the appropriate pore size and porosity have a significant influence on the biological function of cells [35, 36]. Therefore, we investigated the effects of the three scaffolds on the biological function of BMSCs. The proliferation and chondrogenic differentiation results suggested that the 7% ECM scaffolds provide an optimal microenvironment for BMSC cartilage regeneration.

On the other hand, we need to further improve the biochemical cues of scaffolds and the driving force of MSCs to enhance cartilage regeneration. GDF-5, a member of the TGF-β/BMP superfamily, plays a critical role in AC development and regeneration [21]. In particular, in a recent study, Sun et al. found that exogenous GDF-5 enhanced BMSC chondrogenic differentiation and migration in vitro [24]. Therefore, GDF-5 and ECM were mixed for hybrid printing, and LDM 3D printing significantly protected the activity of GDF-5. First, we confirmed the effect of GDF-5 on BMSC chondrogenic differentiation and further explored its biological mechanism based on previous studies. Next, we investigated the effect of the hybrid scaffold on BMSC migration in vivo and chondrogenic differentiation in vitro. The hybrid scaffold showed significantly enhanced migration and chondrogenic differentiation. In conclusion, we found that the ECM scaffold incorporating GDF-5 can not only provide the necessary microenvironment for cellular defect repair but also recruit more stem cells to the defect area in vivo, which has positive significance for in situ AC repair.

To confirm the effects of the scaffolds on AC repair in situ, we implanted them into articular cartilage defects in rabbits. The postoperative results showed that cartilage regeneration in the ECM/GDF-5 group was significantly better than that in the other two groups (control and ECM groups), and the gap between the ECM/GDF-5 group and normal cartilage in the sham group was minimal. Combining the in vitro and in vivo experimental results, we propose a possible mechanism by which the ECM/GDF-5 hybrid scaffold enhances AC regeneration. GDF-5 in the scaffold was released in large quantities in the early stage, which acted on MSCs in the joint cavity, especially BMSCs released after microfracture, causing them to migrate to the defect area. Then, the stem cells adhered to the hierarchical porous structure and finally differentiated into chondrocytes. Specifically, as the main component of scaffolds, decellularized ECM combined with GDF-5 can provide a good regenerative microenvironment for cell proliferation and differentiation. Finally, during the interaction between the cells and the scaffold, along with the degradation of the scaffold, regenerative cartilage was gradually produced and finally filled the defect area.

Although the above results showed that the ECM/GDF-5 hybrid scaffold constructed by LDM 3D printing significantly improved the quality of AC repair in situ, there are still some limitations that need to be further explored. First, due to its low cost and easy availability, we used ECM made from porcine AC. The biosafety of this heterogeneous ECM in clinical application may need to be further verified. Second, the mechanical properties of scaffolds made of ECM alone are poor. How to improve the mechanical properties without affecting their biocompatibility is the focus of our next work. Finally, we need to study the long-term effects before clinical translation in large mammals, such as sheep. Despite the above limitations, this ECM/GDF-5 scaffold printed with LDM technology still has broad prospects in the field of AC in situ tissue engineering and clinical transformation.

Conclusions

In summary, for the first time, we used ECM alone as a bioink in LDM 3D printing and then successfully fabricated a hierarchical porous ECM scaffold incorporating GDF-5. According to the in vitro and in vivo experimental results, the ECM/GDF-5 scaffolds not only recruited a large number of stem cells to the defect area but also provided an ideal regenerative microenvironment for the MSCs. Finally, animal models have demonstrated that the ECM/GDF-5 scaffolds promote in situ AC repair in vivo, indicating that this scaffold has significant potential for AC in situ regeneration tissue engineering in the future.