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Zhang, et al.
2.12. mRNA expression 2.15. Biochemical analysis

For mRNA expression analysis, cell-scaffold construct We analyzed the contents of collagen and sulfated-
samples were cultured for 7 and 14 days (n = 3). For glycosaminoglycan (sGAG) of samples. Samples were
mRNA extraction, the samples were homogenized and dissolved in papain digestion (125 μL/mL papain, 100 mM
lysed in TRIzol (Yeasen, Shanghai, China) before being EDTA, and 5 mM L-cysteine; Sigma-Aldrich, MO, USA)
centrifuged at 12,000 rpm at 4°C. Subsequently, a cDNA at pH 6.5 at 60°C under rotation for 18 h. Collagen content
reverse transcription kit (Yeasen, Shanghai, China) was was examined by quantifying hydroxyproline concentration.
used to reverse-transcribed total RNA into cDNA. The Briefly, the hydroxyproline content of the solution
expression levels of cartilage-related markers (COL II, was determined by the chloramine-T assay after acidic
type-2 collagen; ACAN, aggrecan; SOX-9, SRY-box hydrolyzation in 38% HCl for 18 h at 110°C. Collagen content
transcription factor 9) and osteogenic-related markers was calculated by assuming a hydroxyproline: collagen
(COL I, type-1 collagen; OCN, osteocalcin; ALP, alkaline ratio of 1:7.69. The total sGAG content was analyzed by
phosphatase; RNUX2, runt-related transcription factor the 1,9-dimethylmethylene blue (DMMB; Sigma-Aldrich,
2) were analyzed by real-time qPCR system (Applied MO, USA) assay. The sGAG and collagen content were
Biosystems, CA, USA). The target mRNA was normalized normalized by dry weight of construct.
to a housekeeping control (glyceraldehyde 3-phosphate
dehydrogenase mRNA) and determined using the ΔΔCt 2.16. Statistical analysis
method. The sequences are listed in Table S1. All data are expressed as the mean ± standard deviation

2.13. In vivo surgical operation (SD), and P < 0.05 was considered statistically significant.
For statistical analysis, intergroup differences were
To determine the osteochondral regeneration effect in calculated by analysis of variance (ANOVA) after testing
vivo, New Zealand white rabbits weighted 2.0 – 3.0 kg for homogeneity of variance. All statistical analysis
were chosen to create osteochondral defect models. After was performed using Statistical Package for the Social
general anesthesia, osteochondral defects (diameter: Sciences (SPSS) version 19.0 software (IBM SPSS
4 mm, depth: 5 mm) were caused on the patellar groove Statistics for Windows, Armonk, NY, USA).
of right knee joints. In the control group (n = 6), the
defect was left blank without material added. In the 3. Results and discussion
pristine-bilayered construct group (PB group) (n = 6), 3.1. Preparation and characterization of DCM/
the constructs without bioactive growth factors were
implanted into the defects. In the GF-bilayered construct SF and DBM/SF bioinks
group (GB group) (n = 6), bilayered constructs containing To estimate the ECM component change, we determined
TGF-β1 and BMP-2 were implanted in the osteochondral the collagen and glycosaminoglycans (GAGs) contents
defect (Figure S2A and B). The rabbits were anesthetized before and after decellularization (Figure S1A and B).
and executed 3 months after operation. A significant loss of collagen and GAGs in DCM and
2.14. Histological and immunohistochemical DBM was observed after decellularization. The reason
analysis behind the reduction of the collagen and GAGs is the
treatment with the enzymes during the trypsinization
For histological assessment, rabbit femurs were fixed and decellularization including deoxyribonuclease ,
overnight in 4% paraformaldehyde at 4°C and then ribonuclease and pepsin [30,48] . Quantification of DNA
decalcified with decalcification solution for about 30 days. content from native ECM (NCM and NBM) and dECM
The decalcified constructs were dehydrated with a graded (DCM and DBM) revealed the significant reduction
series of ethanol and embedded in paraffin for sectioning. (~96%) in the case of dECM as compared to the native
The deparaffinized paraffin sections were stained with ECM before decellularization.
safranin O and Masson’s trichrome, and imaged with an We investigated rheological behavior to explore
Olympus microscope. To further observe the expression flow properties of DCM/SF and DBM/SF bioinks. The
of COL II and OCN, immunohistochemical staining frequency sweep indicated that viscosity of DCM/SF
was performed as we previously described . The ICRS and DBM/SF bioinks decreased in response to linearly
[47]
Visual Histological Assessment Scale was carried out to increasing shear rate (Figure 2A), indicated that the
score the morphology and the degree of metachromatic bioinks exhibited shear-thinning flow behavior, which
staining, respectively. The total score ranged from 0 to are similar to most polymer gels . Furthermore, the G’
[36]
18, including semi-quantitative scales to rate the surface, values exceeded the G’’ values over the whole angular
matrix, cell distribution, viability of the cell population, frequency range (G’ > G’’; Figure 2B), indicating the
subchondral bone, and cartilage mineralization. formation of a typical gel structure .
[49]
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