The Healing Effect of Vitreous Body on Repair of Cartilage Defects of Knee Joint in Dog as an Animal Model

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Article Information:


Group: 2009
Subgroup: Volume 11, Issue 2
Date: April 2009
Type: Original Article
Start Page: 166
End Page: 169

Authors:

  • M Mehriar
  • Poostchi Eye Research Center, Poostchi Street, Shiraz, Fars, Iran
  • Mahnaz Mosallaei
  • Poostchi Eye Research Center, Poostchi Street, Shiraz, Fars, Iran
  • H Ashraf
  • Poostchi Eye Research Center, Poostchi Street, Shiraz, Fars, Iran
  • S Rezasadeh
  • Department of Orthopedic Surgery, Shiraz University of Medical Sciences, Shiraz, Fars, Iran
  • N Azarpira
  • Organ Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Fars, Iran
  • N Tanideh
  • Comparative Research Center,Department of Pharmacology, Shiraz University of Medical Sciences, Shiraz, Fars, Iran
  • D Mehrabani
  • Stem Cell and Transgenic Technology Research Center, and Gastroenterohepatology Research Center,Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Fars, Iran

      Correspondence:

      Affiliation: Poostchi Eye Research Center, Poostchi Street
      City, Province: Shiraz, Fars
      Country: Iran
      Tel: +98-711-2302830
      Fax: +98-711-2302830
      E-mail: mosalla2000@yahoo.com

Abstract:


Background: To investigate the healing effects of vitreous body in repair of cartilage defects of the knee joint in dog as an animal model.

 

Methods: Ten mature cross-bred female dogs weighing 20-35 kg entered our study. The right and left knees underwent a medial parapatellar incision arthrotomy. A cartilage defect was created by a six mm drill to subchondral bone in the right knee and the two borders of the fascia were fixed to the space of the joint defect. The vitreous was provided through a clear corneal incision to completely fill the joint defect. In the left knee, the cartilage defect was left untreated. The dogs were sacrificed after 3 months post-operation and the samples were studied for any sign of repair.

 

Results: The defects of the right knee showed more signs of repair compared to the left knee after 12 weeks. Fibrous and hyaline tissues, new bone and blood vessels formation were significantly more visible in the right knee. Modified histological scoring scale in the right and left knees were 5.8 and 11, respectively.

 

Conclusion: The results imply the promising effect of vitreous body implantation in the healing of a cartilage defect in the knee joint.

Keywords: Vitreous body; Healing; Cartilage defect; Knee Joint; Dog

Manuscript Body:


Introduction

 

A damaged articular cartilage has limited capacity for treatment.1-3 Several methods were introduced to overcome this limitation. One of them was implantation of a synthetic collagen sponge containing recombinant human bone morphogenetic protein-2 which was performed to accelerate this repair process during a 6 month period.4 Subsequently injection of cultured autologous chondrocytes under a periosteal flap was undertaken in animal models for treatment of a cartilage defect. Although this procedure was successful to some extent, the main problem with that procedure was the loss of implanted cells from the cartilage defect.5 Vitreous body as a jelly structure in the eye is a unique tissue in body with a composition similar to the articular cartilage. This similarity is particularly due to the presence of large amounts of type 2 collagen which is the main collagen of the hyaline cartilage.6,7 So it is proposed that vitreous body as a natural substance can be used in part for induction of repair of cartilage defect.6 The present study was carried out to investigate the healing effect of vitreous body in repair of cartilage defects in the knee joints of dog as an animal model.

 

 

Materials and Methods

 

Ten mature cross-bred dogs with a mean age of 5 months and weight of 10 kg were enrolled. Animal selection, experiments, subsequent care and the sacrifice procedure all adhered to the guidelines and were under supervision of Animal Care Committee of Iran Veterinary Organization. All the experiments were carried out under aseptic conditions in the Laboratory Animal Center of Shiraz University of Medical Sciences. The protocol of anesthesia, surgical procedures, postoperative care and sacrifice were identical for all the animals. Anesthesia was induced by intravenous diazepam (10 mg) and sodium thiopental (0.5 mg) and after endotracheal intubation, the animals were maintained on a controlled ventilation by halothane and 100% oxygen. Ringer’s lactate was administered intravenously throughout the operation (6 ml/kg/h). Before the induction of anesthesia, intravenous penicillin streptomycin (500 mg) and post-operatively three doses of this antibiotic were administered to prevent any unwanted infection. The study was approved by the Ethics Committee of Shiraz University of Medical Sciences.

The right knee was considered as the case (group 1) and the left knee as the control group (group 2). The knees underwent a medial parapatellar incision arthrotomy. A cartilage defect was created by a six mm drill to subchondral bone (depth of 1.5 mm). In the right knee, a piece of fascia was prepared through an identical incision in the distal part of the thigh. The two borders of the fascia were fixed to the defective joint surface by 7-0 vicryl sutures. Simultaneously, the vitreous was provided through a clear corneal incision of the right eye of the dog, using a size 8 trephine. The lens delivery was carried out and the jelly structure was transferred, using a loop like device to fill the defective joint space. (Autologous implantation) The whole surface of the defective cartilage was covered by a fascia to hold the vitreous away from the joint space. The medial retinacolum was repaired by a 3-0 vicryl and the skin was stitched by a 3-0 nylon suture. The cartilage defect in the left knee was left untreated to heal spontaneously and the skin was sutured with the same technique as in the right knee joint. The dogs had an unrestrained movement in their cages immediately after recovery from anesthesia and the movement of each lower extremity was clinically evaluated in the site of operation, 1, 3 and 12 weeks after operation. The dogs were sedated 12 weeks after operation using ketamine (10 mg/kg) and the samples were provided by a cut about 2.5 cm above and below each knee joint. The dogs were euthanized with an overdose injection of potassium chloride. The site of cartilage defect was exposed and macroscopically evaluated. The provided samples from the defective site were transferred to a 10% buffered formalin solution and were histhopathologically examined, providing 6 µm thickness sections stained with Hematoxylin and Eeosin dyes. Any sign of tissue repair was recorded semiqauntatively.7 (Table 1). A modified histological scoring scale including 5 categories ranging from 0 to 14 was used while the lower the score, the better the results of healing. The data were analyzed, using SPSS software (version 12, Chicago, IL, USA).  Non-parametric Wilcoxon Signed Rank test was used to compare both groups. A p value <0.05 was considered significant.

 

Table 1: Modified histological grading scale for the repair of cartilage defect7

Cell morphology                           

Hyalin cartilage                            

0

Mostly hyalin cartilage                 

1

Mostly fibrocartilage                     

2

Mostly  non-cartilage                     

3

Non-cartilage                                   

4

Surface Regularity

Smooth                                              

0

Moderate                                             

1

Irregular                                               

2

Severly irregular                            

3

Filling of Defect

>2/3                                                      

0

1/3-2/3                                                  

1

<1/3                                                        

2

Integration

Both edges integrated                              

0

One edge integrated                              

1

Neither edges integrated                           

2

 

 

Results

 

Defects of the right knee joint  (group 1), treated with implantation of the vitreous body showed more signs of repair compared to the left knee (group 2) after 12 weeks (Figure 1). The cartilage defects were partially filled with white or red-brown tissues which were readily distinguishable from the surrounding intact cartilage surface. An acceptable interface was noticed between the repaired tissue and the host cartilage without any signs of disruption in group 1. In group 2, severe depression of the repaired tissue and disruption of the interfaces were detected. Fibrous and hyaline cartilage tissues and new bone and blood vessel formation were significantly more in group 1. Hyaline cartilage was visible as islands in group 1 (Figure 2,3). No non-cartilaginous tissue was demonesterated in this group, while non-cartilaginous tissue was the prominent finding in group 2 without any presence of hyaline cartilage (Figure 4). The results of modified histological scoring scale were 5.8 and 11 in group I and 2, respectively, indicative of a significant improvement in repair of the cartilage  in group 1 (p value=0.02). Moreover, according to each one of the items of the scoring system, the results of group 1 were statistically more considerable than those of group 2, especially in relation to better surface integrity in group 1.

 

 

Fig 1: Fibrous tissue formation in untreated joint (H&Ex400).

 

 

 

Fig 2: Islands of new hyaline cartilage tissue adjacent to normal articular cartilage in the right side of treated joint (H&Ex400).

 

 

Fig 3: New hyaline cartilage formation in treated joint (H&Ex100).

 

 

 

 

Fig 4: The defect in the left side is brown color and deep, but the defect in the right side (treated side) is creamy glistening and the edge with the normal cartilage is not sharp or abrupt.

 

 

Discussion

 

Articular cartilage has a very limited capacity for the regeneration of a damaged cartilage; however, there has been no definite surgical or medical treatment till now to overcome this problem.1,2,8 Vitreous tissue has probably a kind of immune privilege such as aqueous humor confirmed by absence of inflammation after injection of gram negative organisms derivitives.5,9-11 Dogs were more used in this relation as a human model.5 Cartilage defect was considered in a non-weight bearing area of the medial condyle of  the femur to abolish the potential of loading the weight to study the healing effects in the cartilage tissue.3,5 In our study, implantation of the vitreous could considerably increase repair in the hyaline tissues. To our knowledge, this effect has not been reported before except for a report using an autologous chondrocyte transplantation (ACT).5,8,12 The effect of the vitreous body in repair of the cartilage tissue may be partly pertained to the presence of type 2 collagens and hyaluronic acid as a background for migration of proliferated chondrocytes. Moreover, solidification of chitosan-glycerol  in the micro-fractures showed an improvement in the repair of the defects which might, to some extent, be due to an increase in the amount of tissue, the biochemical composition, and the cellular organization.12,13 Since cell loss in the space of defects  was one of the main problems with ACT, authologous blood clot was used only as a scaffold to enhance the adherence of cells.14,15 Therefore, regardless of the especial characteristics of the vitreous body as a natural component resembling hyaline cartilage, its acceptable effects in cartilage repair can be interpreted at least in the context of making tissue adherence and improvement of local tissue composition. The results imply the promising effect of the vitreous body implantation in healing of a cartilage defect in the knee joint.

 

 

Acknowledgement

 

We wish to appreciate the financial support of the study by Office of Vice Chancellor for Research of Shiraz University of Medical Sciences.

 

Conflict of interest: None declared.

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