Engineered mucoperiosteal scaffold for cleft palate
regeneration towards the non-immunogenic transplantation

Ethics

The study was conducted in accordance with ethical standards and principles expressed in the Declaration of Helsinki. Informed consent was obtained from all donors included in the study and approved by the local Ethics Committee of the Bambino Gesù Children’s Hospital.

All animal procedures were fully compliant with Italian (Ministry of Health guidelines, Legislative Decree No. 116/1992) and European Union (Directive No. 86/609/EEC) legislations on animal research.

All experimental protocols were carried out in strict accordance with the approved guidelines of Italian legislation.

Pigs and palates harvesting

All animal procedures were fully compliant with Italian (Ministry of Health guidelines, Legislative Decree No. 116/1992) and European Union (Directive No. 86/609/EEC) legislations on animal research.

The methods were carried out in strict accordance with the approved guidelines.

The mucoperiosteum of the hard palate of six pigs (pig 1–6) obtained from a local abattoir were stored in dry ice in Styrofoam container and transported to the laboratory within 1–2 h of their collection. Randomly selected pieces from each fresh palate were collected for DNA quantification before dry storage at − 80 °C. All the palates were then subjected to the standard decellularization protocol and they were longitudinally divided into two identical hemi-palates, resulting in a total of 12 samples, randomly divided into two groups, the pored (P) and the non-pored (NP), with only those belonging to the P-group being further manipulated for the perforative treatment, as stated above.

Palate decellularization

Each palate was washed in ultrapure water and 2% Penicillin–Streptomycin (P/S, Sigma-Aldrich) for 48 h at 4 °C in static conditions and incubated five times for 4 h at room temperature in 4% sodium deoxycholate (BioXtra ≥ 98.0%—Sigma-Aldrich). Samples were then treated with 2000 Kunitz Unit (KU) of DNase-I (Warthington) in 1 M NaCl for 3 h at and incubated at 37 °C (5×) and then rinsed in ultrapure water and 2% P/S. In order to remove decellularization reagents, samples were washed with increasing percentages of denatured ethanol (ACS Reagent, ≥ 99.8%, without additive—Honeywell) and then rehydrated overnight in ultrapure water. Scaffolds were then stored in Phosphate Buffered Saline (PBS, Sigma-Aldrich) and 1% P/S at 4 °C. Efficiency of decellularization protocol was evaluated by DNA quantification.

Scaffold perforative treatment

After decellularization, each palate was cut longitudinally in order to divide them into two samples for a comparative analysis: one half was left intact, while the other half was subjected to a microscopic patented perforative treatment (patented by Telea Biotech) using the VESALIUS current generator, based on Quantum Molecular Resonance (QMR) technology (property of Telea Electronic Engineering Srl)48, connected to a 300 μm-diameter needle mounted on a 3-axis Cartesian robot (Yamaha model RCX240) handpiece. Procedures were performed in class-II biological safety cabinet under aseptic conditions. The treatment allows to make micropores on biological tissues with adjustable dimensions and densities up to 1000 pores/cm2 without causing burning phenomena or affecting the surrounding ECM structure, thus allowing to significantly increase the available surface for cell attachment49. Its efficacy it had already been demonstrated in a recent esophageal reconstruction study on piglets with the same technique. Pores’ creation and dimensions were analyzed with digital microscope (Vision Engineering model EVO Cam) at different magnifications50.

DNA quantification

Double stranded DNA (dsDNA) was quantified in fresh samples and in both decellularized only and decellularized/pored scaffolds. dsDNA was isolated using the DNeasy Blood and Tissue kit (Qiagen) following manufacturer’s instructions. DNA was quantified by using a BioPhotometer Plus (Eppendorf). Optical densities at 260 nm and 280 nm were used to estimate DNA purity and yield.

Palate recellularization

Cell cultures

MSCs were isolated and cultured as previously described40. Briefly, mononuclear cells were isolated from residual cells of a healthy donor, who donated BM for transplantation at the Bambino Gesù Children’s Hospital, and after obtaining a written informed assent/consent. Cultures were maintained at 37 °C in a humidified atmosphere, containing 5% CO2. After 48-h adhesion, non-adherent cells were removed and culture proceeded with culture medium being replaced twice a week. MSCs were harvested, after reaching ≥ 80% confluence and were propagated at 4 × 103 cells/cm2.

Immunophenotype

MSCs were phenotypically characterized by flow cytometry using different fluorophore-conjugated monoclonal antibodies specific for CD34, CD45, CD73, CD80, CD86, CD90, CD105 and HLA-DR. (BD PharMingen, San Diego, CA). Analysis of cell populations was performed by means of direct immunofluorescence with a FACSCanto flow-cytometer (BD PharMingen) and data were calculated using the FACSDiva software (Tree Star, Inc. Ash-land, OR).

Differentiation capacity

The differentiation potential of MSCs was assessed as previously described40. Osteogenic and adipogenic differentiation were evaluated following calcium and fat droplets deposition via Alizarin Red and Oil Red O (Sigma-Aldrich, St Louis, MO) staining, respectively.

Seeding

Before seeding, part of each scaffold was excised as control. MSCs from passage 2 (P2) and 4 (P4) were harvested, counted and resuspended at a concentration of 1 × 106/ml. Cells were then seeded on each decellularized scaffold (pored or non-pored) immobilized on the bottom of a petri dish, previously washed and re-equilibrated in medium. After 2 h enough medium to cover the seeded scaffolds was added, maintaining the culture for 14 days and performing 3 re-seeding as previously described. Part of each scaffold were excised at day 7 (d7) and at the end of culturing (d14) to be tested as described afterwards.

Ultra structural analysis

Light and fluorescence microscopy

Decellularized scaffold samples, excised pre- and post-seeding (d7 and d14), were fixed in 10% formalin for 24 h and embedded in paraffin. Slices of 2 µm thickness were then cut and placed on positive charged slides and stained either with Dapi or with hematoxylin/eosin. Slides were the analyzed by light microscopy with Eclipse E600 (Nikon) the following days.

Scanning electron microscopy—SEM

Both pre- and post-seeding scaffolds (d7 and d14) were fixed in 1% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in increasing ethanol concentrations (50%, 70%, 80%, 90% and 100% for 10 min each) and hexamethyldisilazane (HDMS)-dried. The specimens were then mounted on aluminum stubs, gold-sputtered by the Agar High Resolution Coater equipment, and analyzed by SUPRA 25 SEM (Zeiss, Germany) microscope.

Molecular analysis

Real-time quantitative reverse transcription PCR (qRT-PCR)

All reverse transcription-quantitative PCR (RT-qPCR) experiments were performed as previously described51. Quantitative RT-PCR was performed in triplicates using inventoried TaqMan Gene expression assays purchased from Applied Biosystems. Relative mRNA levels for genes of interest were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyltransferase (HPRT), TATA-binding protein (TBP) and beta glucuronidase (GUSB) taken as housekeeping genes and calculated by using the 2−ΔΔCt method. Three independent experiments were performed with the ABI 7500 Sequence Detection System Analyzer for RT-qPCR (Applied Biosystems). Results were expressed as mean fold changes induced by post-seeded treatments and comparing d7 and d14 scaffolds. ID codes for TaqMan probes against gene targets analyzed in this work are provided in Table 2.

Table 2 Taqman assays used for RT-qPCR.

Table 2 showed the list of TaqMan primers and probes used for the gene expression assays (Applied Biosystems) in real-time RT-qPCR analysis.

Western blotting

The MSC-seeded scaffolds were lysed in ice-cold lysis buffer (NaCl 150 mM, Tris–HCl 50 mM pH 8, EDTA 2 mM) containing 1% Triton X-100, 0.1% SDS, 1× protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium orthovanadate (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium fluoride (Sigma-Aldrich, St. Louis, MO, USA), and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). The lysate was sonicated for 5 min using a Diagenode Bioruptor Standard Waterbath Sonicator (medium level). Samples were spun down at 13,000×g at 4 °C for 15 min. Supernatant was quantified for protein content using the Bradford method (DC Protein Assay; Bio-Rad, Hercules, CA, USA). Equal amounts of proteins were diluted in Laemmli buffer, boiled and resolved using SDS-PAGE. Primary antibodies anti-COL1A1, anti-SPARC, anti-alpha-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase, GAPDH (Abcam, Cambridge, UK) were incubated overnight and signals were revealed with HRP-conjugated secondary antibodies (Cell Signaling Technology Inc., Danvers, MA, USA) and chemiluminescent substrates (Cyanagen, Bologna, BO, Italy). UVItec Cambridge Alliance was used to detect and quantify the luminescent signal of the protein bands. Expression levels of the target proteins were normalized to those of the housekeeping protein GAPDH (loading control) in each lane. Representative images of WB were cropped for data presentation in the main figure. Full length WB images are shown in Supplementary material.

Statistical analysis

Sample sizes were chosen with adequate power (0.8) according to results of pilot data sets, including our own, which used similar methods. Sample estimation and statistical analyses were performed using SigmaPlot 14 software. Data were first tested for equal variance (Brown-Forsythe) and normality (Shapiro–Wilk test) and the appropriate statistical tests were chosen. The statistical tests used (i.e., Student’s t-test, two-way ANOVA and Bonferroni t-test) are indicated in the main text and in the corresponding figure legends for each experiment. Sample size (n = 3/group) is indicated in the figure legend and represents independent experiments from different animals. Significance in normally distributed samples was analyzed using a two-tailed Student’s t test and two-ways analysis of variance (ANOVA) for post hoc pair wise comparisons including the control group. Post-hoc multiple comparisons were performed with Bonferroni correction. All statistical tests were two-tailed and the level of significance was set at 0.05. Results are shown as mean ± SEM.

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