Recently, the team of Fan Zengjie from the School of Stomatology in Lanzhou University published “3D Printing Hydrogel Scaffolds with Nano-hydroxyapatite Gradient to Effectively Repair Osteochondral Defects in Rats” in Advanced Functional Materials (IF=16.836). Scaffolds with a three-layer structure was the first designed and successfully prepared, accurately mimicking the structure of cartilage, calcified cartilage, and subchondral bone. This research promoted the application of hydrogel scaffolds in the osteochondral tissue engineering. The first corresponding author of the paper is Fan Zengjie, a professor at the School of Stomatology, Lanzhou University, and the first author is Zhang Hui, his academic postgraduate in the class of 2018.

To date, osteochondral defects pose a significant challenge without a satisfactory repair strategy. The complicated synthesis procedures and limited surgery size of engineered grafts inhibit the clinical application of traditional manufacturing techniques. Even with the help of the 3D printing technique, limited osteochondral regeneration in vivo has been achieved so far. One of the main reasons is because the prepared scaffolds for osteochondral repair do not match the real structure of osteochondral tissue, which has a unique gradient structure. In this report, a nano-hydroxyapatite (nHA)/hydrogel composite with a gradient structure that exactly matches the natural cartilage-subchondral bone was prepared via 3D printing. The printed scaffold consists of a pure hydrogel-based top cartilage layer, an interfacial layer (40/60% (w w^-1) nHA/hydrogel) mimicking the calcified cartilage, and a 70/30% (w w^-1) nHA/hydrogel bottom layer mimicking the subchondral bone layer.  

Figure 1. Procedures of 3D printing multi-layered gradient scaffold for OC defect repair in the rat model. (A) Electronic spray device. (a-c) “0% nHA” bio-ink, “40% nHA” bio-ink, and “70% nHA” bio-ink, respectively. (i)-(iii): superficial layer (cartilage), intermediate layer (calcified cartilage), and deep layer (subchondral bone) of the osteochondral region. (nHA: nano-hydroxyapatite; SA: sodium alginate; CA: calcium alginate; AM: acrylamide; PAM: polyacrylamide; BMSCs: bone marrow stromal cells.) 

The preparation process of the scaffold is shown in Figure 2. This process can be divided into three steps: 1) Preparing three kinds of hydrogel inks; 2) 3D printing bio-inks; 3) Photo-crosslinking and Ca²⁺ crosslinking. The most important technical innovation lies in the application of an electronic spray device to control the slow release of Ca²⁺ to prevent precipitation, which makes the printing of the 70/30% (w w^-1) nHA/hydrogel layer with an extremely high filler concentration possible.

Figure 2. Synthesis steps of 3D printing hydrogel scaffold materials of “0% nHA” (A), “40% nHA” (B) and “70% nHA” (C). 

3D printed scaffold not only retained its original shape (Figure 3), but also had a porous structure and high mechanical strength (Figure 4). It is explicit that the precise localization of all hydrogels within the scaffolds presented the interconnectivity, the structural integrity, as well as the desired uniformity of fibers and porosity. Besides, the hydrogel with a higher concentration of nHA possesses smaller pores. A reasonable explanation is that the space of the DN hydrogels is taken up by more nHA particles along with the decrease of SA/AM hydrogel content. It is estimated that the pore size of the gradient scaffolds is in the range of 100–800 nm, and varies depending on the nHA content in each layer. The mechanical tests showed that the tensile strength of gradient group was 75KPa and the compressive strength reached 900KPa. These results indicated that this novel method was suitable for fabricating the natural scaffolds with the precise, complex and well-defined shapes.

Figure 3. Photographs of various 3D printed scaffolds. (A) From left to right in (a) were “0% nHA”, “40% nHA”, “70% nHA” (dyed in red with rhodamine), and “G-nHA”, respectively; (b to e) and (f to i) were the states of removal from the supporting and bending of the corresponding samples in (A). (B) Macroscopic appearance of 3D printed artificial meniscus geometric models based on “0% nHA” (a), “40% nHA” (b), “70% nHA” (c) (dyed in red with rhodamine), and “G-nHA” gel (d), respectively; (d and f) were before and after soaking CaCl2 solution (100 mM). (C) Digital images of 3D printed hydrogel scaffolds before immersing in CaCl2 solution for the tensile testing: top-view images of “0% nHA” (a), “40% nHA” (b), “70% nHA” (c) and “G-nHA” (d) and side-view image of “G-nHA” scaffolds (e). (Scale bars = 5 mm)

 

Figure 4. Characterizations of 3D-printing scaffold materials with various nHA/hydrogel mass ratios. A) FTIR spectra and B) XRD patterns of various samples. The characteristic peaks of PO₄^3- and CO₃^2- groups were marked by rectangles. C) SEM images of all samples. Top-down micrographs (a&d), cross-section of the peripheral region (h&l) and high magnification view of the surfaces (e-g) & (I-k). (a-c): “0% nHA” layer, “40% nHA” layer, and “70% nHA” layer, respectively. D) Stress-strain curves and E) compression strength of pure hydrogel, and nHA/hydrogel composite scaffold group with different concentrations of nHA.

The proliferation assay was conducted to evaluate the cell proliferation rate on the scaffolds using the MTT assay kit and AO/EB staining (Figure 5), where live cells are green and dead cells are red. All the material groups in which few cells were stained with red fluorescence showed no obvious toxicity during the 4-day culture, illustrating that the nHA-containing hydrogels had great biocompatibility and the scaffold preparation process was also bio-safe.

Figure 5. Cell viability and proliferation activity on various scaffolds. A) Proliferation activity of goat TMJ disc cells evaluated by MTT assay after an incubation of 1, 2 and 4 days on each group. The data are expressed as mean ± SD; the error bars represent the SD. (All the **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs the control group; n = 3). B) Images of AO/EB staining when goat TMJ disc cells were co-cultured with different samples for 1, 2 and 4 days. (Scale bars = 100 μm)

To assess the in vivo repair of cartilage defects, a total of 56 male SD rats weighing 200g on average were randomly divided into seven groups. Four rats (8 knee joints) in each group at 6 and 12 weeks were sacrificed by an overdose of chloral hydrate to assess the repair process. To sum up, the BMSCs-loaded “G-nHA” group exhibited the optimal OC defect repair in several respects: 1) boundaries were gradually obscured by the content and texture assembling the adjacent normal tissue; 2) defect coverage increased with the content shifting from basic fibrous tissue, fibrous and chondral mixtures to the final hyaline cartilage-like and bony tissue; 3) defects flattened gradually with the growth of more tissue while caved in with distinct steps in the control/BMSCs group.

 

Figure 6. Regeneration of cartilage-subchondral bone in critical-sized defects at 6 and 12 weeks after implantation. Frontal views (A-G) and (A’-G’) and micro-CT images (H-N) and (H’-N’) revealed that the tissues in “G-nHA+BMSCs” exhibited better defect resurfacing than those in other scaffolds. 3D reconstructed images (H-N) and 2D reconstructions in transaxial view (H’-N’) of the repaired defects at 12 weeks after the operation. (Scale bars = 2 mm; Red squares or rectangles indicate the defect areas; White arrows denote a mixture of incompletely degraded scaffold and bony bone; n=4)

Figure 7. Histological and immunohistochemical analysis of the OC defect area of the grafts in the seven groups stained with HE (a&b), Saf-O/Fast Green (c&d), and COL II (e&f). The regions in Saf-O/Fast Green and IHC staining were similar to those depicted in H&E staining. One can observe that (G) exhibits the best tissue morphology at pre-determined time-points. (A)-(G) represent the blank control group, BMSCs group, “G-nHA” group, “0% nHA+BMSCs” group, “40% nHA+BMSCs” group, “70% nHA+BMSCs” group, and “G-nHA+BMSCs” group, respectively. (Red arrows: tissue demarcation; Black arrows: defect areas left by the degraded scaffold; ×4; n = 3; Scale bars = 400 μm)

In summary, an integrated gradient nHA/hydrogel scaffold was designed with a goal of possessing an increasing gradient of stiffness from the cartilaginous layer to the subchondral bone layer. The results showed the promising application of this construct to be fabricated into complex-shaped OC tissues with a porous microstructure, remarkable mechanical properties, appropriate degradability, and high structural integrity for long periods of cell culture. It was of great importance that the gross evaluation, micro-CT, and histological evaluation confirmed the optimal in vivo repair outcomes of the BMSCs-loaded gradient hydrogel with OC regeneration after 12 weeks of postimplantation. In brief, our results provide an experimental and theoretical basis for the application of the gradient nHA/hydrogel composite in OC repair.