This study sought to evaluate the effects of intra-articular injection of insulin-like growth factor-1 (IGF-1) suspended in hyaluronan (HA) on the cartilage and subchondral cancellous bone repair in osteoarthritis (OA) of the temporomandibular joint (TMJ). Disc perforation was performed bilaterally in rabbit TMJs to induce OA. Four groups of animals ( n = 12) received OA induction only, and either intra-articular HA injection alone, intra-articular IGF-1 injection alone, or a combination of HA and IGF-1 injection. All therapy was begun 4 weeks after OA induction. The animals were killed 12 or 24 weeks after the first injection, for histology and micro-CT examinations. Two additional animals were used as normal controls. Typical cartilage and subchondral cancellous bone lesions were observed in the OA group. No protective effect on cartilage and subchondral cancellous bone was found in the HA or IGF-1 alone groups. Better histological repair and nearly normal micro-architectural properties of the subchondral cancellous bone were observed in the HA + IGF-1 group compared with the HA or IGF-1 alone groups. HA may be used as an effective carrier for intra-articular injection of IGF-1 and the combination of HA/IGF-1 shows promise as a new rational approach to therapy of TMJ OA.
Articular cartilage is a specialized type of connective tissue, and maintenance of articular cartilage depends on a balance of anabolic and catabolic activities. In diseases such as osteoarthritis (OA), cartilage degeneration occurs when catabolic activities exceed anabolic stimuli . A number of growth factors are involved in the deterioration of articular cartilage. Among these factors, insulin-like growth factor-1 (IGF-1) is one of the most important anabolic proteins . IGF-1 has been shown to induce chondrocyte synthesis of proteoglycan and collagen and its importance in the etiology of osteoarthritis is also confirmed . Growth factors are made up of soluble proteins of relatively small molecular mass, and direct in vivo application of IGF-1 leads to its rapid diffusion, denaturation and degradation and limited therapeutic effects. The controlled application of IGF-1 with the help of a carrier may solved this problem and enhance its therapeutic effects.
Different delivery systems may influence the bioactivity and longevity of IGF-1, varying its in vivo effects. The use of natural biomaterials may be an attractive concept for the development of IGF-1 carriers. Hyaluronan (HA) is a naturally derived, linear, high molecular weight protein with viscoelastic properties . It is a major component of synovial fluid and plays an important role in joint motion. HA has unique and excellent physicochemical properties, such as biodegradability, biocompatibility, and non-immunogenicity .
HA has received increasing interest as a potential agent of intervention in OA. In OA, the concentration and molecular weight of synovial fluid HA are decreased. Supplementing exogenous HA in the joint space improves the viscoelastic properties of the synovial fluid, and has been reported to relieve joint pain in patients with OA . HA has beneficial molecular and cellular effects in vitro , such as inhibiting prostaglandin E2 (PGE2) synthesis and protecting against proteoglycan depletion . In cartilage, HA has been shown to suppress cartilage matrix degradation by fibronectin fragments .
Recent studies suggest that HA may be used as an attractive material capable of releasing growth factor in a sustained manner. R adomsky et al. found that a solution of HA can form a viscous gel that acts as a diffusional barrier to maintain fibroblast growth factor (FGF-2) at the injection site, and improves callus formation and the mechanical properties at the osteotomy site . K im et al. documented that acrylated hyaluronic acid loaded with bone morphogenetic protein (BMP-2) and human mesenchymal stem cells are a suitable carrier for cells and growth factors for tissue regeneration .
The authors sought to confirm whether commercially available, injectable HA is an effective carrier for the application of IGF-1, and intra-articular injection of IGF-1 with HA enhances the repair of articular cartilage and subchondral cancellous bone. The effect of IGF-1 suspended in HA on cartilage and subchondral cancellous bone repair in the osteoarthritic temporomandibular joint (TMJ) was evaluated by gross examination, histology, and micro-computed tomography (micro-CT).
Materials and methods
50 skeletally mature New Zealand white rabbits (6 months old, 2.5–3.2 kg) were used in this study. They were given tap water and food ad libitum during the experiment. They were kept in separate cages and allowed to move freely. This study was authorized by the Animal Ethics Committee of the University.
48 rabbits were anaesthetized by intravenous injection of sodium pentobarbital 20 mg/kg and sodium ketamine hydrochloride 4 mg/kg. In brief, the hair of the skin covering the TMJ was shaved, and the skin was cleaned with betadine solution. The skin of the posterior area of the eye was cut and the right TMJ was exposed over the zygomatico-squamosal suture. One-third of the disc in the anterior and lateral regions of the joint was resected with a scalpel. The articular capsule and skin were closed independently in layers with 5–0 nylon sutures. The contralateral joint received identical surgery. Piperacillin was administered intramuscularly twice daily for 3 days. All rabbits were returned to their cages after the operation and were allowed to move freely. The animals were given a normal diet for the remainder of the experiment. Two additional animals were used as normal controls and their TMJs remained intact without any treatment.
Sodium hyaluronate was obtained commercially from Freda Co., Shandong, China. The molecular weight is between 1.5 and 2.5 × l0 6 MW and the concentration of hyaluronic acid was 10 mg/ml. The product is available in 2-ml vials. In order to increase viscosity each vial was stored at 4 °C until use. 1 μg of IGF-1 (human recombinant, Sigma Chemical Co., St. Louis, MO, USA) was mixed with 2 ml sodium hyaluronate solution to form HA (10 mg/ml) + IGF-1(0.5 μg/ml) formulation. In the IGF-1 alone group, 1 μg of IGF-1 was mixed with 2 ml physiological saline (0.5 μg/ml).
4 weeks after OA induction as described above, the location of the joint was identified by palpating its lateral aspect with the mouth opened and closed several times, and a syringe with a 26 G needle was inserted into the upper joint cavity. The rabbits were randomly divided into the following four groups ( n = 12): OA group, OA induction only; HA group, OA induction and single intra-articular injection of HA (0.5 mg HA/50 μl,); IGF-1 group, OA induction and single intra-articular injection of IGF-1 (25 ng IGF-1/50 μl); HA/IGF-1 group, OA induction and single intra-articular injection of IGF-1 suspended in HA (0.5 mg HA + 25 ng IGF-1/50 μl). Six animals in each group were killed at 12 and 24 weeks after the first intra-articular injection and the specimens were harvested for gross, histology, and micro-CT examinations.
The mandibular condyles were dissected, fixed in 10% buffered formalin, decalcified with 5% ethylenediamine tetraacetic acid (EDTA) and embedded in paraffin. Sections 5 μm thick were cut for hematoxylin and eosin (HE) and safranine O staining. For each specimen, three randomly selected sections in each joint were evaluated for the severity of OA by two independent blinded observers using the criteria shown in Table 1 .
|Mild increase or decrease
|Moderate increase or decrease
|Severe increase or decrease
|Arrangement of chondrocytes
|Appearance of clustering
|Mild surface irregularities
|Moderate surface irregularities
|Severe surface irregularities
For analysis with micro-CT, subchondral cancellous bone was defined as the cancellous bone region 0.5 mm beneath the calcified cartilage–bone junction. The region of interest was selected as the cancellous bone region 0.5 mm beneath the calcified cartilage–bone junction and 3 mm distal. The subchondral cancellous bone was scanned by a micro-CT 80 scanner (Scanco Medical, Bassersdorf, Switzerland) with a resolution of 2048 × 2048 pixels and at a fixed threshold of 80 according to the manufacturer’s protocol. The parameters for analysis included bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and bone surface density (BSD).
All animals tolerated the injection well, and there was no evidence of local inflammation, immobilisation, or unloading of the joint resulting from the treatment. All animals maintained their body weight during the experiment.
At 12 weeks after injection, the mandibular condyles from the OA group had highly irregular surfaces and most of the condyles exhibited small osteophytes along the dorsal part of the condyle ( Fig. 1 A and B). At 24 weeks after injection, besides severe surface irregularity and osteophytes formation, the joints had a marked reduction in the joint space, indicating fibrous ankylosis between the condyle and articular fossa. The gross appearance of the mandibular condyles from the HA group or IGF-1 group were similar to those from the OA group at both observation time points. In general, the mandibular condyles in the HA/IGF-1 group 12 or 24 weeks after injection showed round, smooth, consistent surfaces that resembled normal cartilage ( Fig. 1 C and D).
Histology of the normal condyle is shown in Fig. 2 ; a well-organized fibrocartilage covering the surface of condyle was observed. Tissue from the OA group displayed the histological features of OA with loss of safranin O staining and hypocellularity of chondrocytes. A number of specimens demonstrated markedly decreased chondrocytes in cartilage. Cartilage from the IGF-1 or HA groups was abnormal and reflected similar osteoarthritic changes to the OA group ( Fig. 3 A–C). Cartilage from the HA/IGF-1 group exhibited less severe cartilage changes with intact articular surfaces, mild amounts of chondrocyte cloning and slight increase in safranin O staining ( Fig. 3 D, E and F). No significant differences in the histological scores were observed between the OA, HA alone and IGF-1 alone groups, whereas the HA + IGF-1 group had a significantly lower score than the other three groups ( P < 0.05, Fig. 4 ).