Matrilin3/TGFβ3 gelatin microparticles promote
chondrogenesis, prevent hypertrophy, and induce paracrine release
in MSC spheroid for disc regeneration

Reagents and materials

Gelatin from porcine skin, paraffin oil, SPAN80, EDC, and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (USA). Glutaraldehyde and acetone were purchased from Daejung Chemicals and Metals (South Korea). TGF-β3 and matrilin-3 were purchased from R&D System (USA). TGF-β3 antibody was purchased from Proteintech (USA) and FITC-labeled matrilin-3 antibody was purchased from Biorbyt (USA). An Alexa Fluor 594 antibody labeling kit was obtained from Life Technologies (USA). StemFit 3D® was purchased from Microfit (South Korea). All culture media were purchased from Hyclone (GE Healthcare, UT, USA). A cell counting kit-8 (CCK-8) assay was purchased from Dojindo (Japan). Trizol reagent was purchased from Qiagen (Hilden, Germany), while the reverse transcription kit and SYBR® Green were purchased from Takara (Japan).

Fabrication of matrilin-3- and TGF-β3-loaded gelatin microparticles (MATN3–GMP and TGF–β3-GMP)

Matrilin-3 and TGF-β3-loaded GMPs were prepared in accordance with our previous studies via a water-in-oil emulsion method. First, 5 g of gelatin type A (Sigma–Aldrich) bloom 175 was dissolved in 50 mL of distilled deionized water at 40 °C, 700 rpm for 1 h until no visible gelatin particles were present. The gelatin solution was transferred drop-by-drop (1 mL/minute) via a syringe into a flask containing 400 mL of olive oil with 1% SPAN80 for 30 min at RT with an additional 30 min of stirring at 4 °C, 500 rpm to create a water-to-oil emulsion. After 1 h, 40 mM glutaraldehyde was added to the GMP solutions for crosslinking. The stirring was continued for 2 h more. The GMPs were then vacuum-filtered in a Buchner’s funnel and washed with cold acetone. The residual oil was washed by incubating the microparticles in 400 mL of 0.1% Tween-80 solution. The microparticles were finally washed with distilled water, lyophilized, and used for further characterization and experiments.

The proteins matrilin-3 and TGF-β3 were conjugated onto the microparticle via EDC/NHS conjugation. The microparticles were first incubated in an activation buffer containing 50 mM MES hydrate, pH 6.0, for 2 h with constant agitation. They were then collected via centrifugation, counted, and incubated in conjugation buffer (50 mM MES buffer, 4.8 mM EDC, and 48 mM NHS) containing protein solutions at a concentration of 100 ng of matrilin-3 and 100 ng of TGF-β3 per 7500 GMPs at 4 °C overnight. Upon conjugation, the microparticles were washed twice with MES buffer and PBS. Finally, the growth factor-conjugated GMPs were lyophilized and ethylene oxide (EO)-sterilized prior to use.

Characterization and immunostaining of the GMP microparticles

Dry GMPs were weighed and resuspended in distilled water in preweighted Eppendorf tubes for 30 min and 1 h with constant agitation. Upon swelling, the tubes were spun at 5000 rpm, and all the excess water was removed. The swollen particles were weighed and recorded. The swelling ratio (q) was calculated using the equation \(q = \frac{{swollen\;MP\;weight\;\left( {W_s} \right)}}{{dry\;MP\;weight\;\left( {W_d} \right)}}\). Finally, the water content was determined with the equation \(\left( {\frac{{W_s – W_d}}{{W_s}}} \right) \ast 100.\)

For the degradation assay, 5 mg of wet microparticles were resuspended with 10 µg/mL of collagenase I in PBS in Eppendorf tubes. The tubes were spun at specific time points, and 200 µL of the supernatant was collected and replaced with the same volume of new media. The collected samples were then tested for the presence of proteins with the BCA assay, and the measured protein concentration was recorded and plotted against the length of incubation. Microparticles resuspended in PBS served as the negative control.

To determine the presence of conjugated proteins, immunostaining was performed. The primary antibody for TGF-β3 (Proteintech, USA) was first labeled with Alexa Fluor 594 with an antibody-labeling kit in accordance with the manufacturer’s protocol. Conjugated matrilin-3 was visualized using FITC-labeled anti-matrilin-3 antibodies. The labeled antibodies were then added to the GMP solution and incubated for 30 min. The stained GMPs were observed with a fluorescence microscope (CKX41; OLYMPUS, Japan).

Maintenance of adipose-derived mesenchymal stem cells (ASCs)

With approval from the Institutional Review Board of the Dongguk University Hospital Ethics Committee (IRB no. DUIRB-202006-09), adipose and chondrogenic tissues were obtained via manual isolation from the knee of donor patients from the Dongguk University Hospital, who provided written informed consent. ASCs and DCs were then isolated from the fat and cartilage tissues, respectively, in accordance with the protocol described in our previous study39.

ASCs and DCs were maintained in DMEM media (Gibco, BRL) supplemented with 10% FBS, and 1% penicillin/streptomycin (Gibco, BRL). The cells were incubated in humidified air with 5% CO2 at 37 °C. The media was changed every other day and the cells were passaged at 80% confluency. ASCs limited to passage 3 were used for all experiments, while DCs were subcultured until passage 6 prior to use. Morphological and molecular characterization of ASCs was done via FACS analysis, while multilineage differentiation potentials were verified via specific staining procedures (Supplementary Figure 2).

Formation, incorporation of GMP–MATN3/TGF-β3, and chondrogenic differentiation of ASC spheroids

Uniformly sized ASC cell spheroids were generated using StemFit 3D (MicroFIT) cell culture microwells in accordance with the manufacturer’s instructions. Briefly, the microwells were washed with 70% ethanol, washed with 1× PBS twice, and finally coated with an anti-adherent rinsing solution (Stem Cell) for 15 min before use. Trypsin-dissociated ASCs were then seeded into the microwells at a concentration of 5.0 × 105 cells/mL and maintained in low-glucose DMEM media at 37 °C in a 5% CO2 incubator for 24 h. The spheroids were then collected and transferred into ultra-low-attachment culture plates and incubated in chondrogenic differentiation media (high‐glucose DMEM medium supplemented with 1% insulin–transferrin–selenium, 50 μg/mL ascorbic acid, and 100 nM dexamethasone without TGF-β1). GMP–MATN3-, GMP–TGFβ3-, or GMP–MIX-incorporated cell spheroids were generated via the same method. A cell suspension containing 5.0 × 105 cells/mL with GMP–MATN3 (100 ng of matrilin-3 per 7500 GMPs), GMP–TGF-β3 (100 ng of TGF-β3 per 7500 GMPs), or GMP–MIX (100 ng of matrilin-3 and 100 ng of TGF-β3 per 7500 GMPs) was seeded into StemFit 3D (MicroFIT) cell culture microwells, incubated for 24 h for spheroid formation, and subsequently transferred to a low-attachment culture plate for chondrogenic differentiation.

Chondrogenic differentiation of ASC spheroids was performed for a total of 14 days, with sampling and analysis for chondrogenesis performed on days 7 and 14. Chondrogenesis and hypertrophy were measured via real-time quantitative PCR, western blot, staining, and immunohistochemical analyses (Supplementary Fig. 1).

Coculture of ASC spheroids with DCs

At 7 and 14 days post chondrogenic differentiation, the ASC spheroids were cocultured with DCs. Trypsin-dissociated DCs (passage 5) were cultured on a 6-well cell insert at a concentration of 7.5 × 104 cells/well on top of the differentiated spheroids. Both ASC spheroids and DCs were incubated in high‐glucose DMEM medium supplemented with 1% of insulin–transferrin–selenium, 50 μg/mL of ascorbic acid, and 100 nM dexamethasone. Differentiated spheroids served as the source of chondrogenic factors. The coculture was maintained for seven days with media replacement every two days.

Total RNA extraction and gene expression analysis with qRT-PCR

RNA samples from ASC spheroids containing GMP only, GMP–MATN3, GMP–TGF-β3, and GMP–MIX were extracted and subjected to real-time PCR analysis at days 7 and 14 of differentiation. Total RNA was isolated via the conventional Trizol method (Gibco Invitrogen, Carlsbad, CA). One microgram of the total RNA was then used for cDNA synthesis using TOPscriptTM cDNA synthesis kit (Enzynomics, Daejeon, Korea). PCR analyses were performed with Power Syber Green PCR Master Mix using 1:10 dilutions of the cDNA samples and 10 pmol of the gene-specific primers. The samples were subjected to the following PCR conditions: repeated denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and extension at 72 °C for 30 s. The sequences of the primers are listed in Table 1.

Table 1 Primers pairs used for real-time quantitative PCR.

Protein expression analysis via western blotting

Total protein was extracted from the cells by incubating the ASC spheroids in 1x RIPA lysis buffer. The protein concentrations were quantified using the BCA assay, and 20 ng/µL protein samples were separated via denaturing PAGE gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat skimmed milk (BD Difco) in TBST solution (Tris-buffered saline Triton ×100) for 1 h and subsequently incubated with primary antibodies (1:500 dilutions for SOX9, ACAN, COL2A, CD44, COLX, and RUNX2) in TBST solution with 5% BSA overnight at 4oC. Membranes were then washed, reblocked, and incubated with secondary antibodies (1:2500 dilutions for goat anti-mouse-HRP, goat anti-rabbit-HRP) in TBST solution with 5% nonfat skimmed milk for 1 hr. Detection of immunoreactive bands was performed using E-select (AmershamTM, Italy), and images were visualized using Bio-Rad Image Lab software. The antibodies used for western blot analyses are listed in Table 2. All blots derived from the same experiment were processed in parallel.

Table 2 Antibodies used for Western Blot analysis.

Staining and immunohistochemical analysis of differentiated ASC spheroids

Chondrogenic differentiated ASC spheroids were analyzed via Alcian blue staining and immunohistochemistry using conventional protocols. First, cell spheroids were collected and washed with 1 × PBS 2 or 3 times, followed by incubation and fixation in 4% PFA overnight. Fixed-cell spheroids were then embedded in Histogel solution and dehydrated in a series of ethanol solutions with increasing concentrations, cleared in xylene solution, and finally embedded in paraffin. The paraffin blocks were then sectioned using a microtome set for 5 µm per section.

For Alcian blue staining, sections of cell spheroids were rehydrated in ethanol with decreasing concentrations and finally washed with PBS. The tissue sections were incubated in 1% Alcian blue solution (in 3% acetic acid, pH 2.5) for 1 h and were subsequently washed with tap water, dehydrated in a series of increasing ethanol solutions, cleared with xylene, mounted with Acrylamount (StatLab, TX), and visualized under a bright-field microscope.

For collagen-2 immunostaining, sections of cell spheroids were rehydrated in a series of ethanol solutions with decreasing concentrations and finally washed with PBS. The tissue sections were incubated in 5% BSA in PBS blocking solution for 1 h and subsequently incubated in collagen-2 primary antibody (1:500) overnight, washed with 1× PBS, and then further incubated in HRP-conjugated secondary antibody for 2 h. The immunostaining was visualized using liquid DAB + substrate chromogen detection (DAKO). Staining intensities of the spheroid sections were digitally quantified using the ImageJ software.

Analysis of cytokine secretion

Cytokine secretion in the coculture conditions was analyzed via a Custom Sandwich-based Antibody Array (RayBiotech Inc., Norcross, GA, USA). In this analysis, the media from the coculture setups were collected, centrifuged to remove cells and cell debris, and processed according to the manufacturer’s protocol. Immunoreactivity was detected and visualized using a ChemiDocTM XRS + detection system (BIORAD iNtRON Biotechnology). The signal densities for each protein were semiquantitatively analyzed using Image Lab software (Bio-Rad) and were normalized to the positive controls for each sample.

In vivo tail IVD degeneration model via nucleus pulposus needle puncture

All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Dongguk University (IACUC-2018-021-2). Briefly, 8-week-old SD rats were raised under specific pathogen-free (SPF) conditions with a light/dark cycle of 12 h, 55–65% humidity, a temperature of 24 ± 3 °C, and free access to food and water. The SD rats were anesthetized by intramuscular injection of xylazine (Rompun, 10 mg/kg; Bayer, Seoul, Korea) with tiletamine hydrochloride/zolazepam hydrochloride (Zoletil, 50 mg/kg; Virbac Laboratories, Carros, France). Tail IVD degeneration was performed by needle puncture after partial subcutaneous incision, as described in previous reports24,40,41. In brief, the tail was prepared for aseptic surgery with povidone iodine and alcohol. A 4- to 5-cm subcutaneous incision was performed on Co6/Co7/Co8/Co9 of rat-tail skin. Subsequently, the IVDs of Co6/Co7, Co7/Co8, and Co8/Co9 were punctured by 5 mm via a 21-G spinal needle, and the needle was turned 360° inside the IVD.

A week after DDD induction, each group was injected with 20 μL (5 × 105 cells with GMPs in PBS) in the center of the IVD via a 21-G spinal needle. The SD rats (n = 3/3 sites per rat) were divided into five groups (n = 9 sites/group) to confirm the synergistic effect of MATN3- and TGF-β3-loaded GMPs in ASC spheroids. After 8 weeks of the initial puncture (seven weeks after implantation), all rats were sacrificed in a CO2 gas chamber, and the rat-tail tissues were collected for MRI analysis and tissue staining.

MRI analysis of IVDs

The MRI analysis was performed as previously described [1]. Briefly, a 4.7-T MRI spectrometer (Bruker Biospec 47/40) and custom MR coil were used for coronal and sagittal T2 MRI with the following settings: time to repetition, 3200 ms; time to echo, 130 ms; matrix, 320 (horizontal) × 320 (vertical); field of view, 120°; and slice thickness, 2 mm, with 0.2-mm spacing between each slice. T2 MRI of all groups was performed to evaluate disc water-content changes 8 weeks after degeneration induction and injection. The T2 signal intensity of the AF and NP of all disks were measured via ImageJ software.

Statistical analysis

The data are representative of three independent experiments with each experiment done in triplicate. Error bars denote the means ± standard error of mean (SEM) and the differences with p values < 0.05 were considered statistically significant (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare the mean values among groups. Individual data points and p-values for significance are indicated in the graph.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Read original article here.