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Research: I. Ishii, H. Mizuta, A. Sei, J. Hirose, S. Kudo, and Y. Hiraki Healing of full-thickness defects of the articular cartilage in rabbits using fibroblast growth factor-2 and a fibrin sealant J Bone Joint Surg Br 2007; 89-B: 693-700 [Abstract] [Full text] [PDF]

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Yuji Hiraki, Issei Ishii, Hiroshi Mizuta, Satoshi Kudo   (22 August 2007)

Healing of full-thickness defects of the articular cartilage in rabbits

Benedict A Rogers, NJ Little   (4 July 2007)

Authors' reply: 22 August 2007
Yuji Hiraki, Professor Institute for Frontier Medical Sciences, Kyoto University, Issei Ishii, Hiroshi Mizuta, Satoshi Kudo Send letter to journal: Re: Authors' reply: hiraki{at}frontier.kyoto-u.ac.jp Yuji Hiraki, et al.	Sir, We appreciate the interest from Dr Rogers in our work. 1. He raised a question regarding the accuracy of cell counting using a haemocytometer. In a monolayer culture system of Balb/c3T3 fibroblasts, as used in this study, the method for the determination of cell numbers by direct cell counting using a haemocytometer is a well-established, simplified technique. A homogenous cell suspension can be conditioned by easily lifting off the cells from the culture plate by trypsin treatment. Therefore, the technique has also been used to verify the mitogenic activity of growth factors in many recent reports.1,2 Meanwhile, given the accurate calibration of DNA content in a cell and the quantitative extraction of DNA from sample tissue, fluorescent DNA assay essentially gives a result equivalent to that from the direct cell counting method. It was difficult in cartilage explants to get cell isolated from the organisation because of large quantities of extracellular matrices such as collagen and proteoglycan; hence direct cell counting was not feasible and so indirect estimation of cell numbers by fluorescent DNA measurement was used as a reliable technique. 2. Dr Rogers also made a point about whether the histological response to FGF-2 demonstrated in this study represented a characteristic chondral reaction, or was this a unique response of chondrocytes in high load-bearing lesion? We used a full-thickness articular cartilage defect model, made in the centre of a trochlea of a rabbit knee (i.e., a weight-bearing lesion) in our series of studies of FGF-2-induced chondrogenic repair of full-thickness articular cartilage defects. Further investigations bearing in mind that the effects of mechanical stimulus such as articulated motion3 and weight-bearing4 using another experimental model or study design was needed to reveal similarities or differences in the FGF-2-induced chondrogenic repair responses between a weight-bearing and a non-weight-bearing lesion. We expect that Dr Rogers is interested in the histological response to FGF-2 in this study, taking into consideration the situation of donor site mobility of a newly formed chondral defect at the site in which a chondral graft was harvested, or the stimulation of chondral repair at the site of cultured chondrocytes-implanted articular cartilage defects. However, we want to point out that the action of FGF-2 examined by this study is not that against the chondrocytes. We think that the results of this study do not just apply to autologous chondrocyte implantation. The repair of a full-thickness articular cartilage defect basically depends on the mobilisation and differentiation of chondroprogenitor cells from the bone marrow cavity. Stimulation of active expansion and maintenance of the chondroprogenitor cell population by stimulation of the mobilisation and recruitment of undifferentiated mesenchymal cells from bone marrow is the main regenerative repair mechanism in rabbit full-thickness articular cartilage defects. This was shown in our earlier studies quoted in this article. The effect of FGF-2 appears after a time as short as one-day exposure. This suggests that FGF-2 acts on the early events in the repair processes for a full-thickness articular cartilage defect, compared with the effects of weight-bearing on the function of chondrocytes such as the expression of a differentiated phenotype, matrix synthesis and/or organisation. When differentiated chondrocytes were implanted, the possibility was considered that the difference of the site of cell harvest (i.e., the metabolic capacity of chondrocytes such as production and organisation of matrix) might bring differences to the results of repair. In the FGF-2-induced chondrogenic repair process initiated by undifferentiated chondroprogenitor cells, we speculated that a big difference would not occur in the repair result of full-thickness articular cartilage defects between weight-bearing and non- weight-bearing lesions. the load after a reparative response may influence the maturity of a reparative chondral tissue once formed. Y. Hiraki, Professor, I. Ishii, H. Mizuta, S. Kudo, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan. 1. Perets A, Baruch Y, Weisbuch F, et al. Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres. J Biomed Mater Res 2003;65A:489-97. 2. Sahni A, Altland OD, Francis CW. FGF-2 but not FGF-1 binds fibrin and supports prolonged endothelial cell growth. J Thromb Haemost 2003;1:1304-10. 3. Salter RB, Simmons DF, Malcolm BW, et al. The biological effect of continuous passive motion on the healing of full-thickness defects in articular cartilage. J Bone Joint Surg [Am] 1980;62-A:1232-51. 4. Harada Y, Tomita N, Nakajima M, Ikeuchi K, Wakitani S. Effect of low loading and joint immobilization for spontaneous repair of osteochondral defect in the knees of weightless (tail suspension) rats. J Orthop Sci 2005;10:508-14.
Healing of full-thickness defects of the articular cartilage in rabbits 4 July 2007
Benedict A Rogers, Specialist Registrar South West Thames, NJ Little Send letter to journal: Re: Healing of full-thickness defects of the articular cartilage in rabbits benedictrogers{at}hotmail.com Benedict A Rogers, et al.	Sir, We read this paper with interest and we would like to make the following points: 1. The in-vitro analysis of FGF-2 bioactivity was quantified by counting cells, after tryspin digestion, in both the fibrin sealant (FS) and FGF-2 groups with a haemocytometer. Fluorometric DNA assay using a bisbenzimidazole dye has previously been shown to provide a validated, reproducible and accurate measure of cellular density within cartilage explants.1 How accurate is measurement of cellular density by haemocytometer, as employed in this study, in comparison to fluorometric analysis? 2. Significant topographical variation exists in the histology, morphology2,3 and biochemical composition4 of articular cartilage with chondrocytes being intrinsically sensitive to load. This study assessed healing in distal femoral trochlea - a high load-bearing region. Does the histological response to FGF-2 demonstrated in this study represent a characteristic chondral reaction or is this a unique response of chondrocytes in high load-bearing loci? It would be informative to ascertain whether a similar response to FGF-2 was achieved from chondral defects in non load-bearing regions. This would have clinical implications since chondrocytes for autologous chondrocyte implantation are currently harvested from non load-bearing regions. BA ROGERS, MA, MSc, MRCGP, MRCS, Specialist Registrar, NJ LITTLE MSc, MRCS, Specialist Registrar, The Princess Royal Hospital, Haywards Heath, UK. 1. Kim YJ, Sah RL, Doong JY, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168-76. 2. Bjelle A. Content and composition of glycosaminoglycans in human knee joint cartilage. Variation with site and age in adults. Connect Tissue Res 1975;3:141-7. 3. Bullough PG, Yawitz PS, Tafra L, Boskey AL. Topographical variations in the morphology and biochemistry of adult canine tibial plateau articular cartilage. J Orthop Res 1985;3:1-16. 4. Rogers BA, Murphy CL, Cannon SR, Briggs TW. Topographical variation in glycosaminoglycan content in human articular cartilage. J Bone Joint Surg [Br] 2006;88-B:1670-4.