Controlled and local release of growth factors and nutrients from porous scaffolds is important for maintenance of cell survival, proliferation, and promotion of tissue regeneration. The purpose of the present research was to design a controlled release porous collagen-microbead hybrid scaffold with controlled pore structure capable of releasing insulin for application to cartilage tissue regeneration. Collagen-microbead hybrid scaffold was prepared by hybridization of insulin loaded PLGA microbeads with collagen using a freeze-drying technique. The pore structure of the hybrid scaffold was controlled by using preprepared ice particulates having a diameter range of 150–250
Hyaline articular cartilage is composed of abundant chondrocytes and limited progenitor cells sparsely embedded in nonvascular extracellular matrix (ECM). Articular cartilage defects are very difficult to heal due to its limited ability of self-repair and regeneration [
Porous scaffolds prepared from biodegradable polymers have been well studied for their ability to regenerate various types of tissues such as skin, cartilage, and bone [
In this research, we have made an attempt to prepare porous scaffolds with a controlled pore structure and controlled release of insulin as a bioactive 3D culture system for cartilage tissue engineering. The porous scaffold was prepared by spatial localization of insulin loaded PLGA microbeads in a 3D collagen porous scaffold by using a freeze-drying technique. Preprepared ice particulates having a diameter range of 150–250
PLGA (copolymer composition ratio of 50 : 50, weight average molecular weight of 20 kDa, and inherent viscosity of 0.187 to 0.229 dL/g), methylene chloride (CH2Cl2), polyvinyl alcohol (86–90 mol% hydrolysis), recombinant human insulin, hydrochloric acid (HCl), sodium hydroxide (NaOH), absolute ethanol (99.5%), N-hydroxysuccinimide esters (NHS), 25% glutaraldehyde solution, and sodium dihydrogen phosphate (NaH2PO4) were obtained from Wako Pure Chemicals Ltd., Japan. L-cysteine hydrochloride monohydrate (minimum 98%), ethylene diamine tetra acetic acid (EDTA), papain, DNA quantification kit, Dulbecco’s Modified Eagle’s Medium (DMEM), growth supplements, and antibiotics were obtained from Sigma-Aldrich, USA. Phosphate buffer saline (10x, pH = 7.4) was obtained from Nacali Tesque Inc., Japan. Porcine collagen type-1 was obtained from Nitta Gelatin, Japan. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC/EDAC) was obtained from Peptide Institute Inc., Japan. Cellstain Double Staining Kit was obtained from Dojindo Laboratories, Japan. Micro BCA protein assay Kit was obtained from Pierce Biotechnology, USA. All the materials in this study were used as received without further purification. Molecular biology grade milli-Q water from millipore water system (Millipore Corporation, USA) was used for preparation of all the solutions and reagents.
Insulin was microencapsulated in PLGA microbeads using w-o-w double emulsion technique [
The collagen-microbead hybrid porous scaffolds were prepared by a freeze-drying method using preprepared ice particulates of a diameter range of 150–250
The morphology of insulin loaded microbeads and scaffold microstructure was examined using a scanning electron microscope (SEM, JSM-5610, JEOL Ltd., Tokyo, Japan). Freeze-dried microbeads were dispersed over a carbon adhesive mounted over a copper stub and were sputtered with a thin layer of platinum by a sputter-coater (ESC-101, Elionix, Tokyo, Japan) for 500 seconds. The freeze-dried collagen scaffolds were cut into cross-sections and mounted on a carbon adhesive over the SEM stub. The cross-sections were sputter-coated with platinum for 300 seconds. The microbeads and scaffold cross-sections were observed at an acceleration potential of 5 kV and 10 kV, respectively.
10 mg of freeze-dried microbeads was suspended in 1 mL of milli-Q water and sonicated for 30 seconds using an ultrasonic water bath. The samples were analyzed for their average size as well as size distribution profile using a laser diffraction particle size analyzer (SALD 7000, Shimadzu Corporation, Japan). The microbead size was measured for three batches of formulation and the mean average was calculated as mean ± standard deviation (
The confirmation of insulin loading as well as insulin quantification in microbeads was carried out using Micro-BCA Protein Assay Kit. 10 mg of dried insulin incorporated microbeads was dissolved in 1 mL of methylene chloride at RT. Insulin was extracted into 0.01 M HCl under vigorous shaking for 2 minutes in a high speed vortex device (Vortex Genie, Fischer, Pittsburgh, PA) at a setting of 10. The suspension was allowed to settle for 5 minutes at RT and supernatant aqueous phase containing insulin was extracted. 150
Compression test was performed for evaluation of mechanical strength of the prepared scaffolds. The control collagen scaffold and collagen-microbead hybrid scaffold were cut into discs of a dimension of
The control collagen scaffolds and collagen-microbead hybrid scaffolds were used for culture of bovine articular chondrocytes (BAC). The scaffolds were cut into discs of a dimension of
The cell-scaffold constructs after 3 hours and 1 week of cell culture were fixed with 0.01% glutaraldehyde at RT. The fixed constructs were washed with milli-Q, freeze-dried and observed for cell adhesion and distribution using SEM.
Cell viability was evaluated by performing live-dead staining assay using Cellstain Double Staining Kit. After 1 week of cell culture, cell-scaffold constructs were washed with PBS and incubated in 2
The cell proliferation in the scaffolds was evaluated by quantifying the DNA amount in cell-scaffold constructs after 1-, 3-, and 7-day culture period. At each time point, the cell-scaffold constructs were collected, washed, and freeze-dried. The freeze-dried cell-scaffold constructs were digested with papain solution. Papain was dissolved at 400
All data were expressed as the mean ± standard deviation (SD). One-way analysis of variance was performed to reveal significant differences, followed by Tukey’s post hoc test for pairwise comparison. Statistical analysis was executed using Kyplot 2.0 beta 15. The difference was considered significant when the
Human recombinant insulin was microencapsulated in PLGA microbeads using a w-o-w double emulsion technique. Figure
SEM photomicrographs of insulin loaded PLGA microbeads (a) size distribution of PLGA microbeads (b).
Porous scaffolds with controlled pore structures were prepared by using preprepared ice particulates as a porogen material. Figure
SEM photomicrographs of control collagen scaffolds (a), (c) and collagen-microbead hybrid porous scaffolds (b), (d) at low (a), (b) and high (c), (d) magnifications. Yellow arrows represent the integrated insulin loaded PLGA microbeads in porous collagen matrix.
The mechanical strength of the scaffolds was determined using a compression test. Figure
Compressive Young’s modulus of control collagen and collagen-microbead hybrid scaffold. Data represent mean ± SD (
Cumulative insulin releases profile (a), (b) and weight loss profile (c) from free microbeads and hybrid scaffold for 4 weeks.
Degradation of microbeads was studied using weight loss profile (Figure
SEM photomicrographs of free microbeads and collagen-microbead hybrid scaffold after incubation for 2 and 4 weeks. Yellow arrows represent the degraded insulin loaded PLGA microbeads in porous collagen matrix.
Control collagen and collagen-microbead hybrid scaffolds were cultured with bovine articular chondrocytes. All the scaffolds were seeded with the cell suspension containing the same number of chondrocytes. The seeding efficiencies of control collagen and collagen-microbead hybrid scaffolds were 87.1 ± 1.1% and 87.0 ± 1.4%. Both control and hybrid scaffolds showed high cell seeding efficiencies. No significant difference in the seeding efficiency was observed among the scaffold groups indicating that microbead incorporation did not cause any difference in seeding efficiency. Figures
SEM photomicrographs of the cross-sections of control collagen (a), (b), (e), and (f) and collagen-microbead hybrid (c), (d), (g), and (h) scaffolds after 3 hours ((a)–(d)) and 1 week ((e)–(h)) of chondrocyte culture. Yellow arrows represent the integrated insulin loaded PLGA microbeads in porous collagen matrix and green arrows represent adhered chondrocytes.
Figure
Live and dead staining of internal cross-sections of control collagen scaffold without insulin (a), control collagen scaffold with 100 nM insulin supplemented in medium (b), and collagen-microbead hybrid scaffold (c) after 1 week of chondrocyte culture. Green fluorescence indicates live cells and red fluorescence dots indicate dead cells.
Figure
DNA amount in the cell/scaffold constructs of control collagen scaffold, control collagen scaffold supplemented with 100 nM insulin, and collagen-microbead hybrid scaffold after 1, 3, and 7 days of chondrocyte culture. Data represent mean ± SD (
The study depicted a controlled release approach to promote cell proliferation in 3D porous collagen for cartilage tissue engineering. Owing to the importance of collagen porous scaffolds of controlled pore structure and improved mechanical strength in cartilage tissue regeneration, controlled release function via biodegradable microbeads was additionally introduced in order to improve the regeneration potential of the prepared scaffold. The present approach suggested the possibility to use other bioactive growth factors as nutrients for cell survival in large and thick 3D porous scaffolds for tissue engineering and regenerative medicine.
A controlled release porous collagen-microbead hybrid scaffold having a controlled pore structure was prepared by introduction of insulin loaded PLGA microbeads into porous collagen sponge formed with preprepared ice particulates. The collagen-microbead hybrid scaffold demonstrated a high mechanical strength and a stable release of insulin for 4 weeks. The released insulin demonstrated its effect on cultured chondrocytes for their survival and proliferation. The bioactive hybrid scaffold should be useful for maintenance of prolonged survival and proliferation of cultured chondrocytes for application to cartilage tissue engineering.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to acknowledge the financial support from World Premier International (WPI) Research Centre Initiative, Ministry of Education, Culture, Sports, Science and Technology, Japan.