Tissue Engineering and Regenerative Medicine, Vol. 10, No. 1, pp 1-9 (2013) DOI 10.1007/s13770-013-0361-0
｜Original Article｜
Novel Repair Technique for Articular Cartilage Defect using a Fibrin and Hyaluronic acid Mixture Jae-Deog Jang2, Young-Seok Moon1, Yong-Sik Kim1, Nam-Yong Choi1, Hyun-Su Mok1, Young-Ju Kim1, Asode Ananthram Shetty3, and Seok-Jung Kim1* 1
Department of Orthopedic Surgery, College of Medicine, The Catholic University of Korea 2 Catholic Institute of Cell Therapy, The Catholic University of Korea 3 Canterbury Christ Church University, Faculty of Health and Social Sciences, 30 Pembroke Court, Chatham Maritime, Kent, ME4 4UF, United Kingdom (Received: July 16th, 2012; Revision: August 28th, 2012; Accepted: October 12th, 2012)
Abstract : We evaluated the cartilage repair potential of a hyaluronic acid and fibrin mixture when transplanted into cartilage defects. Circular, articular, cartilage defects 4-mm in diameter were made in the trochlear region in 21 New Zealand white rabbits divided into three groups. The seven rabbits in the control group underwent microfracture (M group), the seven rabbits in the experimental group underwent microfracture with subsequent injection of hyaluronic acid mixed with fibrin (MH group), and seven rabbits in the other experimental group underwent microfracture followed by injection of bone marrow concentrate and hyaluronic acid mixed with fibrin (MBH group). At week 12 following surgery, the cartilage was observed and histologically compared in the three groups. The surface of the newly generated cartilage was very smooth and even, and we noticed that the entire area was completely regenerated in both experimental groups. The control group showed incomplete and irregular cartilage formation in the defect. In histologic scoring, comparison of the MBH group (M= 2.333) and the M group (M= 9.000) differed significantly (P= 0.046). Therefore, injection of a mixture of bone marrow concentrate, hyaluronic acid and fibrin to treat articular cartilage defects of the knee appears to be an effective method of cartilage regeneration. Key words: hyaluronic acid, cartilage, fibrin, bone marrow concentrate, knee
used for chondromalacia or for small cartilaginous lesions and using traditional surgical methods, the need for a simplified technique continues to increase. Since the 1990s, microfracture has been widely used to treat articular cartilage defects. Numerous successful results with relatively small articular cartilage defects have been reported, although it is recognized that this treatment should be restricted to small defects. Microfracture continues to be used despite the fact that the regenerated cartilage is not hyaline cartilage but mechanically weaker fibrocartilage, as the short-term results have been successful and it is a simple, cost-effective technique.4, 5 There have been efforts to improve the treatment outcomes by facilitating the regeneration of articular cartilage after the microfracture procedure using a bio-scaffold.6 Given this history, following microfracture and using a mixture of hyaluronic acid and fibrin as a scaffold with or without bone marrow concentrate, attempts are being made to investigate the possibility of using this treatment for cartilagin-
1. Introduction For the past several decades, a number of methods have been used for treating damaged articular cartilage, including arthroscopic debridement, microfracture, drilling, osteochondral transplantation, and autologous chondrocyte implantation (ACI). Of these methods, ACI is the most successful for recovering normal articular cartilage. However, ACI is problematic for patients in that they must undergo two surgeries during which part of the normal articular cartilage is extracted and then transplanted following culturing.1, 2 In addition, for the treatment of small articular cartilage defects, higher morbidity has been reported when using cellular transplantation.3 In this regard, as with the treatment methods *Corresponding author Tel: +82-31-820-3654; Fax: +82-31-847-3671 e-mail:
[email protected] (Seok-Jung Kim)
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of the tibia tubercle. Subcutaneous tissue was divided in the line of the skin incision. A medial skin flap was developed to expose the quadriceps tendon, the medial border of the patella, and the medial border of the patellar tendon. A medial parapatellar capsular incision was made, and the patella was dislocated laterally in order to expose the femoral condyle.
ous defects.
2. Materials and Methods 2.1 Experimental Animals Twenty-one twelve-month-old New Zealand white rabbits, each weighing approximately 4 Kg, were kept for one week prior to the experiments, given the same feed and kept under the same conditions. Among them, seven animals were used for the microfracture followed by injection of hyaluronic acid mixed with fibrin (MH), while seven other animals underwent microfracture followed by injection of hyaluronic acid mixed with bone marrow concentrate and fibrin (MBH). The final seven animals constituted the control group (M) of animals with microfracture and undergoing no further treatment. Approval for our animal experiment study was obtained from our Institutional Review Board. This experiment was performed in accordance with the ILAR (Institute of Laboratory Animal Resources) Guide for the Care and Use of Laboratory Animals.
2.2.1 Defect Making and Microfracturing A defect was formed in the subchondral bone by making a lesion 4 mm in diameter (Fig 1A). Using a 23-G needle, three holes were made in the subchondral bone (Fig 1B). 2.2.2 Injection of Hyaluronic Acid and Fibrin Mixture For the injection procedure, two 1-mL syringes and a Yshaped mixing catheter were used. In one syringe, 1 mL of fibrinogen (Greenplast, Green cross Inc., Korea) was inserted, while the other syringe was filled with 0.2 mL of hyaluronic acid (Hyaluma, Hanmi, Seoul, Korea) and 0.8 mL of thrombin (Fig 2A). Hyaluronic acid mixed with fibrin was then slowly injected into the defect area. In order not to overflow the margin of the defect, the position of the defect site was maintained for five minutes. Flexion and extension motion of the knee was then performed three to five times in order to check for any graft failure. The wound was then closed layer by layer.
2.2 Surgical Procedure The rabbits were anesthetized intramuscularly using a mixture of 35 mg/kg of ketamine hydrochloride (Ketamine, Yuhan Co., Korea) and 5 mg/kg of xylazine (Rompun, Bayer Animal Health Co., Korea) and were then placed in a supine position. A longitudinal midline skin incision was made and extended 3 cm above the superior pole of the patella to the level
2.2.3 Injection of Hyaluronic Acid and Bone Marrow Concentrate Mixture The proximal medial surface of the tibia was already exposed due to the incision made for the cartilaginous defect formation surgery. Three mL of bone marrow from the proximal tibia was aspirated using an 18-gauge needle fastened to a 10-mL syringe. Bone marrow aspirates were then combined with the same volume of Dulbecco’s phosphate-buffered saline (DPBS; Gibco, NY, USA). Bone marrow samples were then resuspended and gently layered onto Ficoll-Paque Premium (density 1.077 g/mL; GE Healthcare Bio-Sciences AB, Uppsala, Sweden), after which they were centrifuged at 1153 g for 20 min at 4oC. Nucleated cells were then harvested with blood plasma.
Figure 1. (A) A round hole with a diameter of 4 mm and down to the subchondral bone, was made in the trochlear region (B) Microfracture was performed using a 23-gauge needle.
Figure 2. (A) The mixing procedure for hyaluronic acid and fibrin for the MH group (B) The mixing procedure for hyaluronic acid, fibrin, and bone marrow concentrate for the MBH group.
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thickness of the neo-cartilage was investigated and divided into three grades. Zero points were given if the thickness was at the same level as that of the normal cartilage or more than twothirds that of the surrounding cartilage, and two points were given when the average thickness was less than one-third of that of the surrounding cartilage. Lastly, integration of the donor cartilage with the host adjacent cartilage was graded from 0 when there was no gap between the donor and the host cartilage to two points when there was a complete lack of integration.11
A similar syringe preparation was done as for the 2-2 procedure. In one syringe, a mixture of 0.8 mL of fibrinogen and 0.2 mL of hyaluronic acid was filled, while the other syringe was filled with 0.8 mL of cells with plasma and 0.2 mL of thrombin (Fig 2B). Bone marrow concentrate and hyaluronic acid mixed with fibrin were then slowly injected into the defect area.
2.3 Examination Methods 2.3.1 Gross Appearance Twelve weeks postoperatively, the rabbits were all sacrificed and areas of cartilage defect as well as portions of normal tissue were extracted in order to observe the transitional changes. The gross appearance of the newly generated cartilage was evaluated by comparing it with the normal tissue.7-9
2.3.3 Immunohistochemistry Immunohistochemistry was performed on the same serial sections used in the histochemical staining. Antibodies used in the immunohistochemistry were IH11 for collagen type I (abcam, Cambridge Science Park, UK) and CIIC1 for collagen type II (abcam, Cambridge Science Park, UK). IH11 and CIIC1 were cultured separately at 10% DMEM, and the culture media were separated by centrifuging. Dot-blot analysis was then performed to determine the concentration of an approximate antibody by titration so as to be able to use it as the primary antibody. A universal antibody of the elite ABC kit (Vector, Burlingame, UK) was used as a secondary antibody to induce a secondary antibody reaction, and the DAB reagent (Vector, Burlingame, UK) was used to generate the color reaction.
2.3.2 Histochemical Staining Neo-cartilage and normal cartilage were extracted from each rabbit in each group and were fixed using 3.7% neutral buffered formalin (Sigma, Poole, UK). The cartilage samples were then immersed and decalcified in the decalcifying solution diluted with formic acid: nitric acid:H2O=25:5:70. After decalcification, they were dipped into and neutralized in 5% sodium sulfite solution (Sigma, Poole, UK) and were then rinsed with water. They were then immersed for one hour in each of 50, 60, 70, 80, 90, and 95% ethanol solutions as well as in absolute ethanol so as to be gradually dehydrated. After dehydration, they were immersed in xylene (Duksan, Korea) and were then infiltrated with paraffin so they could be cut into blocks. Sections were cut from the block using a microtome (Leica, Wetzlar, Germany) in order to perform histochemical staining. The sections were then stained with hematoxylin and eosin in order to be able to observe the tissue morphology and with toluidine blue and safranin O in order to check the collagen of the cartilage as well as the GAG content.10 A total of five factors, i.e. cell morphology, matrix staining, surface regularity, thickness of the neo-cartilage, and integrity of the graft with host, were observed, and each cartilage area in the surgical area was evaluated regarding these five features by assigning it a score ranging from 0 to 14 points and representing a modification of that described by Pineda et al. (Table 1).11 First, the cell morphology was graded from 0 (normal cartilage) to four points (no cartilaginous tissue). Next, matrix-staining or the degree of metachromatic staining with toluidine blue was graded from 0 (for tissue that was normal) to three points (no metachromatic staining). Third, the smoothness of the surface of the cartilage was assessed and divided into four grades with 0 points if more than three-quarters of the surface was smooth and three points if less than one-quarter of it was smooth. The
2.3.4 Electron Microscopy 2.3.4.1 Microstructures in the Mixture of Bone Marrow Concentrate and Hyaluronic Acid Mixed with Fibrin A prepared mixture of bone marrow concentrate and hyaluronic acid mixed with fibrin was injected onto the 24 wellplate, after which 0.5 µL of cartilage differentiation media was added. The chondrogenic differentiation medium consisted of Dulbecco’s modified, Eagle’s medium-high glucose (DMEMHG, PAA, Austria) containing 10 ng/mL of transforming growth factor beta 3 (TGF-β3), 25 µg/mL ascorbic acid, 25 µM ascorbic acid-2-phosphate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1% (v/v) ITS plus (all from Sigma-Aldrich, St. Louis, MO, USA). The 24 well-plate was incubated for five days, and the mixture was evaluated using electron microscopy. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the microstructural morphologies seen in hyalurnic acid and in the fibrin mixture with bone marrow concentrate. The mixture was fixed overnight in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M of phosphate buffer, was then washed in 0.1 M of phosphate buffer, and finally post-fixed with 1% osmium tetroxide in the same buffer for 1 hr. The mixture was dehydrated using a graded ethyl alcohol series and acetone. The
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Figure 3. Photographs of the MBH (A-C), MH (D-F), and M(G-I) groups.
mixture was then embedded in Epon 812 (epoxy resin) for TEM processing. Ultra-thin sections (70~80 nm) were obtained using an ultramicrotome (Leica Ultracut UCT, Germany), were double-stained with uranyl acetate/lead citrate, and examined using a JEM 1010 unit (JEOL, Tokyo, Japan) at 60 kV. After dehydration, the specimens were transferred to hexametyldisilazane (HMDS) and were allowed to air dry for SEM processing. All samples were coated with gold using a sputter coater and were examined using a JSM-5410LV unit (JEOL, JAPAN). The SEM was operated at an accelerating voltage of 15 kV.
Table 1. Histologic grading scale for the cartilage defects*. Category
3. Results 3.1 Gross Appearance In all groups, there was a lack of tissue or adhesion constriction. There were also no findings suggestive of synovitis. Twelve weeks postoperatively, in the hyaluronic acid and bone marrow concentrate treatment group (MBH group), each cartilage defect was completely filled with neocartilage, the cartilage surface was very uniform, and there was a smooth connection with normal cartilage (Fig 3A-C). The cartilage color was also uniform. In the hyaluronic acid treatment group (MH group), the cartilage defects were nearly completely filled with neocartilage (Fig 3D-F), although the surfaces showed slight irregularity. In the control group (M group), the cartilage
Points
Cell morphology Hyaline cartilage Mostly hyaline cartilage Mostly fibrocartilage Mostly non-cartilage Non-cartilage only
0 1 2 3 4
Matrix-staining (metachromasia) Normal (compared with adjacent host cartilage) Slightly reduced Markedly reduced No metachromatic stain
0 1 2 3
Surface regularity† Smooth (>3/4) Moderate (>1/2-3/4) Irregular (1/4-1/2) Severely irregular (2/3 1/3-2/3
