Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Summary

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-β1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

Abstract

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-β1) via bioaffinity. The JBNm was assembled at a TGF-β1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-β1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-β1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-β1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

Introduction

Scaffolds in tissue engineering play a vital role in providing structural support for cell attachment and subsequent tissue development¹. Typically, conventional tissue constructs without any scaffolding rely on the cell culture environment and added growth factors to mediate cell differentiation. Furthermore, this addition of bioactive molecules into scaffolds is often the preferred approach in guiding cell differentiation and function^2,3. Some scaffolds can mimic the biochemical microenvironment of native tissues independently, while others can directly influence cell functions vi....

Protocol

1. Synthesis of JBNts

1. Prepare the JBNt monomer utilizing previously published methods, involving the synthesis of a variety of compounds¹².
2. Purify crude JBNt monomer after it has been synthesized with high-performance liquid chromatography (HPLC) using a reverse-phase column. Use solvent A: 100% water, solvent B: 100% acetonitrile, and solvent C: HCl water solution with pH = 1. Use a flow rate of 3 mL/min. Collect the largest peak obtained in the HPLC at 7.2 min.

2. Fabrication for JBNt/Matn1/TGF-β1 (Video 1)

NOTE: V....

Results

Following protocol, JBNts were successfully synthesized and characterized with UV-Vis absorption and TEM. The JBNm is an injectable solid scaffold that undergoes a rapid biomimetic process. After JBNts were added to a mixture of TGF-β1/matrilin-3 solution in a physiological environment, a solid white-mesh scaffold was formed indicating the successful assembly of JBNm, as seen in Figure 1. This was demonstrated in the characterization methods.

Under physiological c.......

Discussion

The goal of this study is to develop a biomimetic scaffold platform, the JBNm, to overcome the limitations of conventional tissue constructs that rely on cell culture environments to mediate cell differentiation. The JBNm is a layer-by-layer structure scaffold for a self-sustainable cartilage tissue construct. The innovative design is based on novel DNA-inspired nanomaterials, the JBNts. The JBNm, composed of JBNts³⁰, TGF-β1, and matrilin-3, is assembled through a novel layer-by-layer techniq.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 % Normal Goat Serum	Thermo Fisher	50062Z	Agent used to block nonspecific antibody binding actions during staining.
24-well plate	Corning	07-200-740	24-well plate used for comparative cell culture.
384-Well Black Untreated Plate	Thermo Fisher	262260	384-well plate used for absorption measurements.
8-well chambered coverglass	Thermo Fisher	155409PK	8-well coverglass used for comparative cell culture.
96-well flat bottom	Corning	07-200-91	96-well plate used for comparative cell culture.
96-Well Plate non- treated	Thermo Fisher	260895	96-well plate used for comparative cell culture and analysis.
Agarose Gel	Sigma-Aldrich	A9539	Hydrogel used for cell culture.
Agarose Gel	Sigma Aldrich	A9539	Hydrogel used as an environment for cell culture.
Alexa Fluor Microscale Protein Labeling Kit	Thermo Fisher	A30006 (488) and A30007 (555)	Fluorescent dye used to label proteins.
Anti-Collagen X Antibody	Thermo Fisher	41-9771-82	Antibody used to stain collagen-X.
Bio-Rad PCR Machine	Bio-Rad		Equipment used to perform PCR on samples.
C28/I2 Chondrocyte Cell Line			Cells used to analyze proliferative abilities of various samples.
Cell Counting Kit 8	Milipore Sigma	96992	Cell proliferation assay.
Cell Profiler	Broad Institute		Software used to analyze cell images.
Cryostat Microtome			Equipment used to produce thin segments of samples for use in staining and microscopy.
DAPI	Invitrogen	D1306	Blue fluorescent stain that binds to adenine-thymine DNA regions.
Disposable cuvettes	FISHER Scientific	14-955-128	Container used for spectrophotometry.
DMEM Cell Culture Medium	Thermo Fisher	10566032	Media used to support cellular growth.
Fetal Bovine Serum	GIBCO	A4766801	Serum used in cell culture medium to support cell growth.
Fluoromount-G Mounting Medium	Thermo Fisher	00-4958-02	Solution used to mount slides for immunostaining.
Formaldehyde			Compound used to fix samples prior to microtoming.
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody	Thermo Fisher	A16110	Antibody used for protein staining.
Human Mesenchymal Stem Cells	LONZA	PT-2501	Cells used to analyze differentiative abilities of various samples.
Human Mesenchymal Stem Chondrogenic Medium	LONZA	PT-3003	Cell medium used to promote chondrogenic differentiation.
ImageJ	National Institutes of Health		Image analysis software used in conjunction with microscopy.
itaq Universal SYBR Green One-Step Kit	BioRad	1725150	Kit used for PCR.
Janus-base nanotubes (JBNts)			Nanotube made from synthetic nucleobases to act as cell scaffolding tool.
LaB6 20-120 kV Transmission Electronic Microscope	Tecnai		Equipment used to perform transmission electron microscopy on a sample.
MATLAB	MathWorks		Statistical software used for modeling and data analysis.
Matrilin-3	Fisher Scientific	3017MN050	Structural protein used as adhesion sites for chondrocytes.
NanoDrop Spectrophotometer	Thermo Fisher		Equipment used to measure absorption values of a sample.
Nikon A1R Spectral Confocal Microscope	Nikon	A1R HD25	Confocal microscope used to analyze samples.
Number 1.5 Chamber Coverglass	Thermo Fisher	152250	Environment for sterile cell culture and imaging.
Optimal Cutting Temperature Compound Reagent			Compound used to embed cells prior to microtoming.
Paraformaldehyde	Thermo Scientific	AAJ19943K2	Compound used to fix cells.
PDC-32G Plasma Cleaner	Harrick Plasma		Cleaner used to prepare grids prior to transmission electron microscopy.
penicillin-streptomycin	GIBCO	15-140-148	Antibiotic agent used to discourage bacterial growth during cell culture.
Phosphate Buffered Saline	Thermo Fisher	10010023	Solution used to wash cell medium and act as a buffer during experimentation.
Rhodamine-phalloidin	Invitrogen	R415	F-Actin red fluorescent dye.
Rneasy Plant Mini Kit	QIAGEN	74904	Kit used to filter and homogenize samples during RNA extraction.
Sucrose Solution			Solution used to process samples prior to microtoming.
TGF beta-1 Human ELISA Kit	Invitrogen	BMS249-4	Assay kit used to determine the presence of TGF-β1 in a sample.
TGF-β1	PEPROTECH	100-21C	Growth factor used for the stimulation of chondrogenic differentiation and proliferation.
Triton-X	Invitrogen	HFH10	Compound used to lyse cells not fixed during staining process.
TRIzol Reagent	Thermo Fisher	15596026	Reagent used to isolate RNA.
Zetasizer Nano ZS	Malvern Panalytical		Equipment used to measure zeta-potential values of a sample.