Icariin-conditioned serum engineered with hyaluronic acid promote repair of articular cartilage defects in rabbit knees

New Zealand White rabbits were obtained from Academy of military medical science in Beijing. Rabbits (4 weeks) were used for isolation of primary chondrocytes, and adult rabbits (12 weeks) were used for drug conditioned serum (DCS) preparation and cartilage defect treatment. The rabbits were kept in special cages in a specific pathogen free (SPF) environment, allowing free movement and free access to food and drink. All procedures were performed in accordance with NIH guidelines for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, revised 1996) and the research protocol was approved by Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC20170026).

Primary chondrocytes were isolated from the articular cartilage of New Zealand White rabbits (four-week-old; Institute of military medical science; China). Briefly, the rabbits were sacrificed and cartilage tissue specimens were obtained from knee and shoulder joints. Fragments were tailored to approximately 1x1x1 mm, washed with cold phosphate buffer saline (PBS), and digested with 0.2% collagenase type II (Invitrogen, Carlsbab, CA, USA) for 4 h. Slices were then digested overnight at 37 °C with 0.025% collagenase type II in PBS on a magnetic stirrer. Isolated cells were suspended in dulbecco’s modified eagle medium (DMEM)/F-12 containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, cultured at 37 °C in a humidified atmosphere of 5% CO₂. Chondrocytes corresponding to passage 3 or below were used for experiments.

Rabbits were treated with ICA (Lot: 160602, Shanghai ronghe, China) to prepare drug-conditioned serum for further administration on chondrocytes and cartilage defects. The equivalent dose of ICA on rabbits calculated according to humans is 0.47 g/kg. Twenty rabbits were randomly divided into four groups, including control, low, middle and high dose groups, which were administered with normal saline, ICA 0.94 g/kg, 2.35 g/kg, 4.70 g/kg at the volume of 10 mL/kg, respectively. Rabbits were gavaged with the different solutions everyday for 1 week. 2 h after the last administration, rabbits were anesthetized with 1 g/kg urethane and blood was sterilely drawn from common carotid artery. Then the blood was centrifuged at 2000 g for 10 min and the supernatants were filtered under 0.22 μm strainer. The DCS was prepared as freeze-dried powder by FD5–2.5 Freeze Dryer (SIM, Newark, USA) and could be freely soluble in normal saline or sodium hyaluronate injection.

Cell proliferation rates were determined using a methyl thiazolyl tetrazolium (MTT, Amresco, Solon, OH, USA) method. Articular chondrocytes cultured in 96-well plates at 8 × 10⁴ cells/mL were treated with 10% DCS of different concentrations in DMEM/F12 for 72 h. Then culture supernatants were replaced with DMEM/F12 and incubated with 10 μL 0.5% MTT solution for 4 h at 37 °C. The absorbance of the blue formazan derivative was measured at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, CA, USA). The experiment was replicated independently for three times.

The amounts of GAG released from chondrocytes were measured using a 1, 9- dimethylmethylene blue (DMMB, Sigma) assay. Articular chondrocytes were plated in 24-well plates at 8 × 10⁴ cells/mL and treated with 10% DCS of different concentrations in DMEM/F12 for 72 h. The absorbance of cell lysates and chondroitin sulfate was detected at the wavelength of 525 nm by a microplate reader. The release of GAG contents were calculated according to the standard curve of chondroitin sulfate. The total DNA content was determined by a PicoGreen DNA kit (Invitrogen, Carlsbab, CA, USA) according to the manufacturer’s protocol and a fluorescence spectrophotometer (F-7000, Hiatachi, Japan) was used for the measurement. The contents of glycosaminoglycan (GAG) were normalized to the DNA content (μg GAG/μg DNA). Each experiment was replicated independently for three times.

Twenty-four male New Zealand White rabbits of mean weight 2.9 kg (range 2.5–3.3 kg) were used for in vivo experiments. Animals were anesthetized by intramuscular injection of 30–40 mg/kg ketamine and fixed in supine position for surgery. A critical-sized defect, 4 mm diameter full-thickness osteochondral defect was created using a surgical drill on the load-bearing area medial femoral condyle to a depth of 3 mm [7]. Penicillin was intramuscularly injected to each rabbit at the dose of 40,000 U/d for 3 days following the operation. All animals were carefully evaluated during the first 24 h after operation.

Rabbits were randomly divided into four groups, including normal saline (NS), ICS (low dose freeze-dried DCS diluted by NS), HA and ICS + HA (low dose freeze-dried DCS diluted by HA) groups. At the beginning of the third week, rabbits in different groups were treated with intra-articular injection of 0.5 mL NS, ICS, HA and ICS + HA in the right knee joint, respectively. All animals were intra-articularly injected of normal saline in the left knee joint. No animals were observed to be infected throughout the experimental period. Rabbits were provided with individual cages and allowed to move freely after surgery. All rabbits were euthanized by an intravenous injection of 120 mg/kg of sodium pentobarbital (MTC Pharmaceuticals, Ontario, Canada) 12 weeks after surgery. The joints of each rabbits from all groups were harvested, and the reparative results were evaluated by macroscopic examination and histological analysis.

The repaired knee joints were retrieved from the hind limbs of the rabbits. The gross appearances of cartilage defects were photographed. The surface characteristics of the regenerated tissue and their integration with the surrounding tissue were blindly examined using the International Cartilage Repair Society (ICRS) macroscopic assessment scale as shown in Table 1 [8, 9].

For cultured chondrocytes, 2 × 10⁴ cells were seeded on slides per well of a 6-well plate and incubated for 2 days. Then chondrocytes were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Toluidine blue staining were performed for characterization of chondrocytes. For characterization of collagen II expression of chondrocytes, cells were treated with HRP-conjugated anti-collagenII primary antibody (1/400 diluted, Bioss, Beijing, China) and visualized with a diaminobenzidine peroxidase substrate kit (Beyotime, Haimen, China). Stained cells were detected with an inverted fluorescent microscope (Ti-U, Nikon, Japan).

After gross examination, repaired knee joints were fixed, decalcified and dehydrated for further embedding. Hematoxylin and eosin (H&E) staining, toluidine blue and safranin O staining were performed for morphological evaluation. For determination of collagen II expression, tissue sections were treated the same with cells. Images were obtained using optical microscopy (YS-100; Nikon; Japan).

Primary chondrocytes were isolated from knee and shoulder joints of rabbits and represented as polygonal or triangular shape with round or oval nucleus (Fig. 1a). At the same time, chondrocytes were characterized by toluidine blue staining and immunohistology of collagen II. The characteristic zonal structure of chondrocytes was embedded in matrix containing high amounts of proteoglycans. This was evidenced by toluidine blue staining (Fig. 1b). Further characteristic properties of chondrocytes are a strong collagen II staining, which was confirmed by positive tan - brown granulates in cytoplasma (Fig. 1c, d).

Chondrocytes were incubated with DCS of different concentrations and the proliferation rates were assessed by MTT assay. After being treated with DCS for 72 h, cells in serum groups exhibited strong proliferation capacity compared with control (P < 0.001, Fig. 2a). ICA-L (low dose icariin conditioned serum), ICA-M (middle dose icariin conditioned serum) and ICA-H (high dose icariin conditioned serum) groups showed much higher proliferation rates when compared with Blank-Serum group (P < 0.001, Fig. 2a). Specifically, chondrocytes in ICA-L group presented strongest growth ability (P < 0.001, Fig. 2a). The GAG is one of main components of cartilage extracellular matrix and the GAG/DNA ratio can be considered as the marker of chondrogenic proliferation. Cells in Blank-Serum, ICA-L and ICA-M groups showed more GAG secretion compared with control (P<0.001, Fig. 2b). In the similar manner with cell proliferation rates, chondrocytes in ICA-L group have the most amount of GAG production (P < 0.001, Fig. 2b). Therefore, DCS prepared with ICA of low concentration was applied in the further in vivo experiment.

HA is a widely used drug for the treatment of osteoarthritis of the knee. As DCS of ICA-L were confirmed to be effective on the proliferation of chondrocytes in vitro, it was applied for the treatment of rabbit articular cartilage defect together with HA. After surgery for 12 weeks, the knee joints of each rabbits were harvested and evaluated by gross morphology examination, histological and immunohistochemical evaluation, as well as semi-quantitative histological scoring analysis. The general morphology observation revealed that the defects were incompletely filled with uneven neo-tissue in 12 weeks in NS group. And the surface of femoral condyle cartilage was not intact, with the exposure of subchondral bone. However, the defect in ICS + HA group was almost repaired and the cartilage surface looked milky and smooth without obvious adhesion (Fig. 3a). The poor degradation of cartilage also exhibited good recovery of femoral condyle. In addition, the defect area was analyzed histologically using the ICRS classification of cartilage regeneration. The ICRS macroscopic scores revealed that the treatment groups acquired significantly high scores when compared with NS group (P < 0.05, Fig. 3b). Among those, ICS + HA group showed much better repair of cartilage defect than other treatment groups (P < 0.05, Fig. 3b). The higher ICRS scores in ICS + HA group indicated better recovery of cartilage defect.

After 12 weeks of healing, H&E staining were performed on the articular joint samples to assess subchondral bone regeneration. In NS group, the regenerated tissues were thinner than treatment groups, with very few hyaline cartilage cells, suggesting inferior tissue integration. However, ICS group and HA group showed more regenerated cartilage than NS group. The repaired tissues in ICS + HA group were totally integrated with surrounding native cartilage. Toluidine blue staining for chondrocytes showed more hyaline cartilage, but few fibrous cartilage in ICS + HA group than other groups. Moreover, the strong Safranin-O staining for GAG, immunehistochemical staining for collagen II also demonstrated that the newly formed tissue was predominantly hyaline cartilage, the same as native cartilage (Fig. 4). The above results confirmed that ICS combined with HA could significantly promote the cartilage defect repair and increase the neoformation of cartilage.