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. 2017 May 20;9(5):182.
doi: 10.3390/polym9050182.

Osteochondral Regeneration Induced by TGF-β Loaded Photo Cross-Linked Hyaluronic Acid Hydrogel Infiltrated in Fused Deposition-Manufactured Composite Scaffold of Hydroxyapatite and Poly (Ethylene Glycol)-Block-Poly(ε-Caprolactone)

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Osteochondral Regeneration Induced by TGF-β Loaded Photo Cross-Linked Hyaluronic Acid Hydrogel Infiltrated in Fused Deposition-Manufactured Composite Scaffold of Hydroxyapatite and Poly (Ethylene Glycol)-Block-Poly(ε-Caprolactone)

Yi-Ho Hsieh et al. Polymers (Basel). .
Free PMC article

Abstract

The aim of this study was to report the fabrication of porous scaffolds with pre-designed internal pores using a fused deposition modeling (FDM) method. Polycaprolactone (PCL) is a suitable material for the FDM method due to the fact it can be melted and has adequate flexural modulus and strength to be formed into a filament. In our study, the filaments of methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) having terminal groups of carboxylic acid were deposited layer by layer. Raw materials having a weight ratio of hydroxyapatite (HAp) to polymer of 1:2 was used for FDM. To promote cell adhesion, amino groups of the Arg-Gly-Asp(RGD) peptide were condensed with the carboxylic groups on the surface of the fabricated scaffold. Then the scaffold was infiltrated with hydrogel of glycidyl methacrylate hyaluronic acid loading with 10 ng/mL of TGF-β1 and photo cross-linked on the top of the scaffolds. Serious tests of mechanical and biological properties were performed in vitro. HAp was found to significantly increase the compressive strength of the porous scaffolds. Among three orientations of the filaments, the lay down pattern 0°/90° scaffolds exhibited the highest compressive strength. Fluorescent staining of the cytoskeleton found that the osteoblast-like cells and stem cells well spread on RGD-modified PEG-PCL film indicating a favorable surface for the proliferation of cells. An in vivo test was performed on rabbit knee. The histological sections indicated that the bone and cartilage defects produced in the knees were fully healed 12 weeks after the implantation of the TGF-β1 loaded hydrogel and scaffolds, and regenerated cartilage was hyaline cartilage as indicated by alcian blue and periodic acid-schiff double staining.

Keywords: cartilage; fused deposition manufacturing; hydrogel; osteoarthritis; poly(ε-caprolactone); scaffold.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Synthesis scheme of the mPEG-PCL-COOH diblock copolymer.
Figure 2
Figure 2
The structure of (A) lay-down patterns 0°/90°(90ECR/90ECR); (B) staggered 0°/90°(SECR/SECR); and (C) 0°/90°/+45°/−45° (45ECR/45ECR) scaffold designs.
Figure 3
Figure 3
The illustration of animal model. Control group: create a bony defect 3 mm in diameter and 8 mm in depth without scaffold implantation. Experimental group: scaffolds and light-curing gel containing TGF-beta 1 were implanted into the defect, and cross-linked with 365 nm ultraviolet light.
Figure 4
Figure 4
The FTIR spectra of mPEG-PCL and mPEG-PCL-COOH, respectively.
Figure 5
Figure 5
1H NMR spectrum of (A) mPEG-PCL and (B) mPEG-PCL-COOH.
Figure 6
Figure 6
Compressive strength of each structure of scaffolds (three groups of samples were tested).
Figure 7
Figure 7
Cytotoxity of (A) scaffolds and (B) GMHA by MTT assay. Error bars represent means with standard deviation.
Figure 8
Figure 8
The DNA content of cell adhesion on 2D materials. Three days after cell culture, DNA detection showed that grafting RGD peptide in the material can effectively enhance cell attachment and proliferation.
Figure 9
Figure 9
The phalloidin staining of the F-actin and LIVE/DEAD staining of cells on 3D scaffolds after 7, 14, 21, and 28 days of culturing.
Figure 10
Figure 10
Chondrogenic differentiation of MSCs in aggregate culture following transduction. Relative expression of mRNA of (A) type II collagen; (B) aggrecan; and (C) Sox9 of MSCs cultured in vitro for up to 28 days. mRNA expression levels were normalized to the housekeeping gene GAPDH and control group.
Figure 11
Figure 11
Biochemical analysis of MSCs differentiation. (A) sGAG contents of the MSCs after 7, 14, 21, and 28 days of differentiation; (B) DNA contents of the MSCs after 7, 14, 21, and 28 days of differentiation and (C) normalized s-GAG contents with respect to DNA contents.
Figure 12
Figure 12
The histological staining (Alcian blue/PAS staining) and specimen of cartilage sections of rabbit after 12 weeks after implantation. (A) The control group with only bone formation; and (B) the experimental group showed bone and hyalin cartilage regeneration (arrow means defect); (C) The specimen of the experimental group showed good cartilage and bone regeneration on the surface layer and residual un-degraded scaffold in the deep layer.

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