Healing of Osteochondral Defects Implanted with Biomimetic Scaffolds of Poly(ε-Caprolactone)/Hydroxyapatite and Glycidyl-Methacrylate-Modified Hyaluronic Acid in a Minipig

Articular cartilage is a structure lack of vascular distribution. Once the cartilage is injured or diseased, it is unable to regenerate by itself. Surgical treatments do not effectively heal defects in articular cartilage. Tissue engineering is the most potential solution to this problem. In this study, methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) and hydroxyapatite at a weight ratio of 2:1 were mixed via fused deposition modeling (FDM) layer by layer to form a solid scaffold. The scaffolds were further infiltrated with glycidyl methacrylate hyaluronic acid loading with 10 ng/mL of Transforming Growth Factor-β1 and photo cross-linked on top of the scaffolds. An in vivo test was performed on the knees of Lanyu miniature pigs for a period of 12 months. The healing process of the osteochondral defects was followed by computer tomography (CT). The defect was fully covered with regenerated tissues in the control pig, while different tissues were grown in the defect of knee of the experimental pig. In the gross anatomy of the cross section, the scaffold remained in the subchondral location, while surface cartilage was regenerated. The cross section of the knees of both the control and experimental pigs were subjected to hematoxylin and eosin staining. The cartilage of the knee in the experimental pig was partially matured, e.g., few chondrocyte cells were enclosed in the lacunae. In the knee of the control pig, the defect was fully grown with fibrocartilage. In another in vivo experiment in a rabbit and a pig, the composite of the TGF-β1-loaded hydrogel and scaffolds was found to regenerate hyaline cartilage. However, scaffolds that remain in the subchondral lesion potentially delay the healing process. Therefore, the structural design of the scaffold should be reconsidered to match the regeneration process of both cartilage and subchondral bone.


GPC Analysis
Gel Permeation Chromatography (GPC 270,Viscotek,Malvern,UK) analysis was performed for the synthesized polymers to determine the number average molecular weight (Mn), the weight average molecular weight (Mw) and polydispersity index (PDI). Using tetrahydrofuran (THF) as the solvent.
Molecular weight and molecular weight distribution were characterized by GPC and 1 H NMR. As mentioned in our previous study, the molecular weight and polydispersity (PDI, Mw/Mn) of mPEG-PCL-COOH polymer is 9514.33 ± 389.70 (Da) and 1.19 ± 0.01.

1 H NMR Spectra
The organic chemical components of the polymer were determined by nuclear magnetic resonance spectrophotometry (500 MHz, Bruker, MA, USA). All samples were measured and recorded as solutions in deuterated chloroform.
The 1 H NMR spectra of mPEG-PCL and mPEG-PCL-COOH were shown in Figure S2. CDCl3 was used as the solvent and the signal of 7.26 ppm was detected. As seen in the spectrum of mPEG-PCL and mPEG-PCL-COOH, the resonance signals at 1.36, 1.65, 2.31, and 4.06 ppm are attributed to the chemical shift of the methylene protons of -(CH2) 3-, -OCCH2-and -CH2OOC-in the PCL unit. The PEG unit shows its characteristic resonance signals at 3.37 and 3.64 ppm, which are associated with CH3-and-CH2CHO-. The spectra of mPEG-PCL-COOH showed the carboxyl group at 2.65 ppm. Figure S2. 1H NMR spectrum of (A) mPEG-PCL and (B) mPEG-PCL-COOH.
The 1 H NMR spectra of GMHA ( Figure S3). The two peaks at 5.8 and 6.5 ppm are associated with the methacrylate groups (c,b), while the methylgroup of HA appears at 2 ppm (a).

TGA Analysis
Thermogravimetric Analysis (TGA) is used to determine the inorganic composition of the materials and to predict their thermal stability. Samples with weights ranging from 10 to 20 mg were put into the furnace and heated from 25 °C to 900 °C. All operations were carried on at the rate of 10 °C/min. From thermogravimetric analysis (TGA), it was observed that the onset of thermal degradation started at about 300 °C, and at 400 °C entire amount of PCL had degraded. In pure HAp, no thermal degradation was observed due to the thermal degradation temperature is in the range between 1360 °C to 1400 °C. As witnessed in the TGA of the scaffolds, the weight percentage that was left in the pan indicated the composition of HAp, which was 35 wt % and comparable to our design.

In Vitro Degradation
To ensure an appropriate bone restoration, the selected material must have a degradation rate that's close to the new bone formation. The in vitro degradation behavior in phosphate buffer solution (PBS) according to the standard protocol ASTM F1635 was investigated. The sterilized RP scaffolds were placed in 100 mL glass container containing 50 mL of PBS and incubated at 37 °C shaking with 50 rpm for three months. The characterization and morphological analysis of the degraded scaffolds were performed After 12 weeks of immersion in PBS, scaffolds exhibited the rough surface with HAp exposure. The image of the scaffolds clearly illustrates the complete cross section of the scaffold matrix is affected by erosion, the white dots on the fracture and erosion surface was noted, too ( Figure S4). According to the phenomenon appeared on the fracture and erosion surfaces, the scaffold underwent both bulk and surface erosion was suspected. After 3 months of degradation, the weight of the scaffold decreased to 20% of its original value. Figure S4. Scanning Electron Microscope Imaging of the scaffold after immersed in PBS solution for 12 weeks.

Cytotoxicity of the Scaffold and Hydrogel
The agar diffusion test on L929 mouse fibroblast cells was used for cytotoxicity analysis. The scaffold (PEG-PCL-RGD) and the hydrogel (GMHA) test materials were placed on the agar, Latex Gloves were used for the material of the positive control group and Teflon was used as a negative control group. Cytotoxicity was observed by measuring the zones of decolorization and evaluating cell lysis under microscope using the established criteria after 24 h.
The cytotoxicity result of agar diffusion test of the scaffold (PEG-PCL-RGD) and the hydrogel (MGHA) are presented in Figure S5. Both the scaffold and the hydrogel showed the decolorization score of 2 and the cell lysis score of 2. A Teflon mold was used as a negative control with no cellular lysis. The latex glove was used as a positive control and resulted in a decolorization score of 4 and cell lysis score of 5. The result indicated that the scaffold (PEG-PCL-RGD) and the hydrogel (MGHA) were low toxicity and cells could grow and proliferation in the environment with the synthetic materials.