3D-bioprinting ready-to-implant anisotropic menisci recapitulate healthy meniscus phenotype and prevent secondary joint degeneration

Objectives: Disruption of anisotropic phenotypes of the meniscus would contribute to OA progression. Exploring phenotype changes of the anisotropic meniscus in joint degeneration would help understand the biologic interaction between the meniscus and OA, and further facilitate the therapeutic strategies of meniscus injury-related joint degeneration. Meanwhile, engineering biomimetic meniscal tissue mimicking the anisotropy of the healthy meniscus remains a challenge. Methods & Results: Meniscal disruption of phenotype anisotropy (PBV growth, cellular phenotype and ECM depositions) was confirmed in OA patient samples. To recapitulate healthy meniscus phenotypes, 3D-bioprinted anisotropic TCM meniscus constructs with PBV growth and regional differential cell and ECM depositions were generated. Transplanted 3D-bioprinted meniscus into rabbit knees recapitulated phenotypes of native healthy meniscus and conferred long-term protection against secondary joint degeneration. Conclusion: 3D-bioprinted TCM meniscus not only restored the anisotropy of native healthy meniscus with PBV infiltration and better shape retention, but better maintained joint function and prevented secondary joint degeneration, which provided a new strategy for the clinical treatment of meniscus injury-related joint degenerative diseases.


In vitro experiments
SMSCs were isolated from synovial biopsies of healthy rabbit knee joints. The SMSC pools were expanded until passage 3 at 37 °C, 5% CO2, and 95% humidity in Dulbecco's modified Eagle medium

Fabrication of scaffolds
Bioprinting rabbit-derived SMSC-laden hydrogels together with biodegradable polymers was conducted for TCM meniscus construction using OPUS system (Novaprint, China). The MSCs suspension (a total of 1 × 10 7 cells) was loaded into the composite hydrogel (Table S1) [1]. TGFβ3 and CTGF μS, with empty μS served as control, were mixed in the cell-laden hydrogel respectively (Table S1)   Twenty-four weeks later, rabbits were sacrificed for further study.

Biomechanical analysis
The biomechanical properties of the explanted scaffold in vitro were assessed using a material-testing machine (Instron 5843, USA).
Pull-out strength for each scaffold-to-native suture rim was calculated and compared. Bidirectional tensile testing was performed with uniaxial tests in the radial and circumferential directions within the samples. In addition, each sample (1-mm thick) was cut into a rectangular shape in the designated regions.
Uniaxial tensile testing was performed on the samples in the circumferential direction as previously described [4]. The samples were tested to failure at a rate of 0.06 mm/s. The elastic modulus was analyzed from the linear portion of the stress-strain curve. In confined compressive testing, a cylindrical sample of 2-mm diameter and 1-mm thickness from the anterior section of the scaffold was used. The creep response of the specimen under a step force (0.02 N) was monitored until reaching equilibrium (defined as slope < 1 × 10 −6 mm/s, for at least 7200 s). The test force was then automatically removed, and the recovery phase was monitored until reaching equilibrium (defined as slope < 1 × 10 −6 mm/s, for at least 3600 s). The aggregate modulus was then calculated using the Mow biphasic theory [5]. Compressive testing in the different anatomic locations (inner or outer regions) in the tissue-engineered meniscus was also performed by nanoindentation. Briefly, samples were isolated from the inner or outer regions of the meniscus tissue in vitro. All indentations were analyzed using a TriboIndenter (Hysitron Inc.) with a 400-mm radius curvature conospherical diamond probe tip. A trapezoidal load function was applied to each indent site (inner or outer region) with loading (10 s), hold (2 s), and unloading (10 s). The operation of the indentations was force-controlled to a maximum indentation depth of 500 nm. The values of reduced modulus and hardness were then calculated.

Statistical analysis
Sample sizes for all quantitative data were determined by power analysis with one-way ANOVA or two-way ANOVA. All statistical data were expressed as means ± SD. Comparisons of differences between construct regions for each stimulus group were used to test the anisotropic properties of the engineered meniscus tissue.
The efficacy of the growth factors in building functional properties within different regions was also tested. One-way ANOVA or two-way ANOVA with Tukey's test was used to analyze the data.
When the two-way ANOVA showed significance (P< 0.05), Tukey's test was applied. Interaction effects were estimated using a general linear model. All data analyses were performed using SPSS statistical software (version 15.0; SPSS Inc.). Values of P< 0.05 were considered statistically significant.  D) Radial tensile modulus. All data are means ± SD (n = 6) and were analyzed by one-way ANOVA with Tukey's test. F) Circumferential tensile modulus. All data are means ± SD (n = 6) and were analyzed by two-way ANOVA with Tukey's test. G-H.
Reduced modulus G) and hardness H). All data are means ± SD (n = 6), and were analyzed by two-way ANOVA with Tukey's test.
Data are presented as averages ± SD and were analyzed by two-way analysis of variance (ANOVA) with Tukey's test. *P< 0.05 between control or TCM group and the native group.