Design, Synthesis, and 3D Fabrication of Novel Nutraceutical Hydrogels to Repair Articular Cartilage
1. Design, Synthesis, and 3D Fabrication of Novel Nutraceutical
Hydrogels to Repair Articular Cartilage
Charles Malcolm Roberson, Roy McReynolds, Amir Hobson, and
Juana Mendenhall, Ph.D.
Morehouse College, Department of Chemistry, Atlanta GA 30314
ABSTRACT
Injury and diseases that affect articular cartilage present a daunting challenge
in tissue engineering applications. During the onset of injury or disease, low-
oxygen environments decrease healthy cartilage cell growth prohibiting the
efficacy of tissue scaffolds in the defective joint. In this study, poly(N-
vinylcaprolactam) [PVCL], hyaluronic acid (HA), and functional nutraceuticals
were synthesized using free radical polymerization and cross-linking
chemistries. PVCL is a biomaterial that undergoes a phase transition (or
separation) in solution in response to temperature called the lower critical
solution temperature (LCST). HA is a polysaccharide that naturally exists in the
knee joint. However, this material diminishes over time in low-oxygen
conditions and eventually loses its ability to provide support for the knee. This
study involved comparative assessments of grafted and cross-linked
copolymers along with the best cross-linking molecules. Grafted copolymer
networks were prepared in aqueous solvents using water-soluble indicators
while cross-linked copolymer systems were prepared using organic solvents.
Using a MakerBot Replicator 2X 3D Printer equipped with syringe-extrusion,
hydrogels were printed to create geometric shapes encapsulated with
cartilage cells to study the effect of cell metabolism under oxidative stress.
Our results show cross-linking hydrogels provide more robust hydrogels for 3D
printing well-defined structures and nutraceuticals provide protection to
cartilage cells when introduced to oxidative stress by improving extracellular
matrix protein synthesis in vitro.
Objective: To report the 3D syringe printing of hydrogels in that can be used to
investigate cell interactions in vitro.
Methods:
Gel Construction
The hydrogels were prepared by combining various concentrations of sodium alginate
with water, forming a gel at 2%, 8%, and 10% using 0.2, 0.3, 0.4, and 0.5 M
concentrations of calcium chloride as an ionic cross-linker.
3D Printing
In order to modify the MakerBot® Replicator 2X 3D Printer to enable printing of
hydrogels, the extruders were removed from the printer, and replaced with syringe
extruders. The extruders were further modified to allow the gels to be printed using the
3D Printer.
To print, the SA hydrogel was placed into the syringe extruder and printed into a shape.
Following each layer of printing, CaCl2 was applied in order to cross-link and solidify the
gel.
Travel Speed 20 mm/s
Z-axis Travel Speed 6 mm/s
Print Speed 28mm/s
Motivation: The goal of this project is to synthesize therapeutic poly N-
vinylcaprolactam (PVCL) hydrogels grafted with naturally occurring nutraceuticals to be
used for cell culture in order to induce cartilage tissue extracellular matrix (ECM)
production in chondrocytes for a potential cure for arthritis.
Methods: Hydrogels were synthesized by free radical polymerization and grafted with
various nutracueticals including catechin, curcurmin, and carvacrol. N-VCL was dissolved
in 18 millipore water in a round bottom flask followed by the addition of one of the
nutraceuticals (Figure. 1). Methacrylated hyaluronic acid was added and the mixture was
put on a reflux condenser in an oil bath and heated to 55 degrees celsius. As the mixture
was heated, the flask was flooded with Argon gas. Once the mixture reached 55 degrees,
VA-057 initiator was added and the reaction was left to run overnight. Finally, the
solution was precipitated in cold acetone and dialyzed for 3 days in 18 millipore water.
Results: Unfortunately it appeared that the meHA used did not dissolve so the reaction
was not completed successfully. This may have been due to the high M.W. of the meHA.
We planned to analyze the hydrogels synthesized using Nuclear Magnetic Resonance
(NMR) spectroscopy however the insolubility of the meHA prevented us from
performing the spectroscopy. Future work includes performing the reaction with meHA
of a lower M.W. to ensure solubility during the reaction and when conducting NMR
spectra.
Figure 5. Synthesis chart of grafted PVCL hydrogels
ACKNOWLEDGEMENTS!
• SMART Biomaterials Lab
• Dr. John Hopps Jr. Defense Research Scholars Program
• Morehouse College Biology Department
• Morehouse College Chemistry Department
Results: Following preparation of the hydrogels, each sample was characterized based
on its mobility and stability. From the results of the gel formation, it appears that it
would be best to proceed making hydrogels from 8% alginate and between 0.3 and 0.5
M CaCl2. These gels showed stability after cross-linking, and we believe they present the
greatest chance for success in future experiments.
COMBINATORIAL SYNTHESIS OF THERAPEUTIC
FUNCTIONAL HYDROGELS
Amir Hobson
3D PRINTING POLY(N-VINYLCAPROLACTAM)-BASED
HYDROGELS USING 3D SYRINGE EXTRUSION TECHNIQUES
Charles Malcolm Roberson
ROBUST CROSS-LINKED HYDROGELS WITH CONTROLLED
THERAPEUTIC DELIVERY
Roy McReynolds
Figure 2. Sodium Alginate Hydrogels
Figure 2. Print Settings for MakerBot 3D Printer
Motivation: The objective of this project is to synthesize and characterize robust
cross-linked hydrogels with controlled therapeutic delivery.
Methods: Hydrogels were synthesized by free radical polymerization and cross
linked with various antioxidants. Poly-N-vinylcaprolactam was dissolved in a
solution of either benzene or ethanol. An antioxidant, such as catechin, carvacrol,
or curcurmin were added to the solution. Methacrylated hyaluronic acid was added
to this solution as well as a crosslinking agent, either Polyethylene glycol or bis-
acrylamide. The solution was then placed into reflux condenser setup, lowered into
an oil bath, and heated to 65°C. As the mixture was heating, the flask was flooded
with Argon gas, to remove moisture from the system. Once the mixture reached 65
degrees, AIBN initiator was added and the reaction was left to run overnight.
Finally, the solution was precipitated in cold hexane and dialyzed for 2-3 days in 18
millipore water.
Results: The samples that were prepared were sterilized using gamma radiation,
and were sent to a lab at Washington State University to be placed into a centrifugal
force bioreactor in order to observe the growth of cells on the samples. Future
work includes testing the hydrogels in a hypoxic chamber to observe how cells will
grow on them.
Alginate (%)
→
[CaCl2] (M) ↓
2 8 10
0
0.2
0.3
0.4
0.5
Figure 1. Modified Syringe Extruder
Figure 4. Picture of 3D Printer while printing
Damage to a knee joint caused by
Osteoarthritis
Potential usage for
this technology
would involve direct
application of
hydrogels into
damaged joints
Figure 3. Setup of 3D Printer
![Design, Synthesis, and 3D Fabrication of Novel Nutraceutical
Hydrogels to Repair Articular Cartilage
Charles Malcolm Roberson, Roy McReynolds, Amir Hobson, and
Juana Mendenhall, Ph.D.
Morehouse College, Department of Chemistry, Atlanta GA 30314
ABSTRACT
Injury and diseases that affect articular cartilage present a daunting challenge
in tissue engineering applications. During the onset of injury or disease, low-
oxygen environments decrease healthy cartilage cell growth prohibiting the
efficacy of tissue scaffolds in the defective joint. In this study, poly(N-
vinylcaprolactam) [PVCL], hyaluronic acid (HA), and functional nutraceuticals
were synthesized using free radical polymerization and cross-linking
chemistries. PVCL is a biomaterial that undergoes a phase transition (or
separation) in solution in response to temperature called the lower critical
solution temperature (LCST). HA is a polysaccharide that naturally exists in the
knee joint. However, this material diminishes over time in low-oxygen
conditions and eventually loses its ability to provide support for the knee. This
study involved comparative assessments of grafted and cross-linked
copolymers along with the best cross-linking molecules. Grafted copolymer
networks were prepared in aqueous solvents using water-soluble indicators
while cross-linked copolymer systems were prepared using organic solvents.
Using a MakerBot Replicator 2X 3D Printer equipped with syringe-extrusion,
hydrogels were printed to create geometric shapes encapsulated with
cartilage cells to study the effect of cell metabolism under oxidative stress.
Our results show cross-linking hydrogels provide more robust hydrogels for 3D
printing well-defined structures and nutraceuticals provide protection to
cartilage cells when introduced to oxidative stress by improving extracellular
matrix protein synthesis in vitro.
Objective: To report the 3D syringe printing of hydrogels in that can be used to
investigate cell interactions in vitro.
Methods:
Gel Construction
The hydrogels were prepared by combining various concentrations of sodium alginate
with water, forming a gel at 2%, 8%, and 10% using 0.2, 0.3, 0.4, and 0.5 M
concentrations of calcium chloride as an ionic cross-linker.
3D Printing
In order to modify the MakerBot® Replicator 2X 3D Printer to enable printing of
hydrogels, the extruders were removed from the printer, and replaced with syringe
extruders. The extruders were further modified to allow the gels to be printed using the
3D Printer.
To print, the SA hydrogel was placed into the syringe extruder and printed into a shape.
Following each layer of printing, CaCl2 was applied in order to cross-link and solidify the
gel.
Travel Speed 20 mm/s
Z-axis Travel Speed 6 mm/s
Print Speed 28mm/s
Motivation: The goal of this project is to synthesize therapeutic poly N-
vinylcaprolactam (PVCL) hydrogels grafted with naturally occurring nutraceuticals to be
used for cell culture in order to induce cartilage tissue extracellular matrix (ECM)
production in chondrocytes for a potential cure for arthritis.
Methods: Hydrogels were synthesized by free radical polymerization and grafted with
various nutracueticals including catechin, curcurmin, and carvacrol. N-VCL was dissolved
in 18 millipore water in a round bottom flask followed by the addition of one of the
nutraceuticals (Figure. 1). Methacrylated hyaluronic acid was added and the mixture was
put on a reflux condenser in an oil bath and heated to 55 degrees celsius. As the mixture
was heated, the flask was flooded with Argon gas. Once the mixture reached 55 degrees,
VA-057 initiator was added and the reaction was left to run overnight. Finally, the
solution was precipitated in cold acetone and dialyzed for 3 days in 18 millipore water.
Results: Unfortunately it appeared that the meHA used did not dissolve so the reaction
was not completed successfully. This may have been due to the high M.W. of the meHA.
We planned to analyze the hydrogels synthesized using Nuclear Magnetic Resonance
(NMR) spectroscopy however the insolubility of the meHA prevented us from
performing the spectroscopy. Future work includes performing the reaction with meHA
of a lower M.W. to ensure solubility during the reaction and when conducting NMR
spectra.
Figure 5. Synthesis chart of grafted PVCL hydrogels
ACKNOWLEDGEMENTS!
• SMART Biomaterials Lab
• Dr. John Hopps Jr. Defense Research Scholars Program
• Morehouse College Biology Department
• Morehouse College Chemistry Department
Results: Following preparation of the hydrogels, each sample was characterized based
on its mobility and stability. From the results of the gel formation, it appears that it
would be best to proceed making hydrogels from 8% alginate and between 0.3 and 0.5
M CaCl2. These gels showed stability after cross-linking, and we believe they present the
greatest chance for success in future experiments.
COMBINATORIAL SYNTHESIS OF THERAPEUTIC
FUNCTIONAL HYDROGELS
Amir Hobson
3D PRINTING POLY(N-VINYLCAPROLACTAM)-BASED
HYDROGELS USING 3D SYRINGE EXTRUSION TECHNIQUES
Charles Malcolm Roberson
ROBUST CROSS-LINKED HYDROGELS WITH CONTROLLED
THERAPEUTIC DELIVERY
Roy McReynolds
Figure 2. Sodium Alginate Hydrogels
Figure 2. Print Settings for MakerBot 3D Printer
Motivation: The objective of this project is to synthesize and characterize robust
cross-linked hydrogels with controlled therapeutic delivery.
Methods: Hydrogels were synthesized by free radical polymerization and cross
linked with various antioxidants. Poly-N-vinylcaprolactam was dissolved in a
solution of either benzene or ethanol. An antioxidant, such as catechin, carvacrol,
or curcurmin were added to the solution. Methacrylated hyaluronic acid was added
to this solution as well as a crosslinking agent, either Polyethylene glycol or bis-
acrylamide. The solution was then placed into reflux condenser setup, lowered into
an oil bath, and heated to 65°C. As the mixture was heating, the flask was flooded
with Argon gas, to remove moisture from the system. Once the mixture reached 65
degrees, AIBN initiator was added and the reaction was left to run overnight.
Finally, the solution was precipitated in cold hexane and dialyzed for 2-3 days in 18
millipore water.
Results: The samples that were prepared were sterilized using gamma radiation,
and were sent to a lab at Washington State University to be placed into a centrifugal
force bioreactor in order to observe the growth of cells on the samples. Future
work includes testing the hydrogels in a hypoxic chamber to observe how cells will
grow on them.
Alginate (%)
→
[CaCl2] (M) ↓
2 8 10
0
0.2
0.3
0.4
0.5
Figure 1. Modified Syringe Extruder
Figure 4. Picture of 3D Printer while printing
Damage to a knee joint caused by
Osteoarthritis
Potential usage for
this technology
would involve direct
application of
hydrogels into
damaged joints
Figure 3. Setup of 3D Printer](https://image.slidesharecdn.com/507d5a8b-b354-4991-9a69-9d776b2bb6e7-160610202813/85/Design-Synthesis-and-3D-Fabrication-of-Novel-Nutraceutical-Hydrogels-to-Repair-Articular-Cartilage-1-320.jpg)
