Molecular and immunohistological characterization of human cartilage two years following autologous cell transplantation.
BACKGROUND
There are only a few studies concerning the cellular, biochemical, and genetic processes that occur during the remodeling of graft tissue after autologous chondrocyte transplantation. The purpose of the present study was to quantify the expression of genes encoding extracellular matrix proteins and regulatory factors that are essential for cell differentiation in cartilage biopsy specimens from patients who had this treatment two years previously.


METHODS
Two cartilage biopsy specimens from each of four patients who had been treated with autologous chondrocyte transplantation and from two multiorgan donors were used. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression of types I, II, and X collagen; aggrecan; cathepsin B; and early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for matrix proteins and regulatory proteins was carried out on paraffin-embedded sections.


RESULTS
Type-I collagen mRNA was expressed in all of the samples evaluated. Type-II collagen was present in autologous chondrocyte transplantation samples but at lower levels than in the controls. Type-X collagen messenger was undetectable. Aggrecan mRNA was present in all of the samples at lower levels than in the controls, while cathepsin-B messenger levels were higher and Egr-1 and Sox-9 mRNAs were expressed at lower levels. The immunohistochemical analysis showed slight positivity for type-I collagen in all of the sections. Type-II collagen was found in all of the samples with positivity confined inside the cells, while the controls displayed a positivity that was diffuse in the extracellular matrix. Cathepsin B was slightly positive in all of the samples, while the controls were negative. Egr-1 protein was particularly evident in the areas negative for type-II collagen. Sox-9 was positive in all samples, with evident localization in the superficial and middle layers.


CONCLUSIONS
In biopsy specimens from autologous chondrocyte transplantation tissue at two years, there is evidence of the formation of new tissue, which displays varying degrees of organization with some fibrous and fibrocartilaginous features. Long-term follow-up investigations are needed to verify whether, once all of the remodeling processes are completed, the newly formed tissue will acquire the more typical features of articular cartilage.
Grigolo
Brunella
B
Laboratorio di Immunologia e Genetica, Istituto di Ricerca Codivilla Putti, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy. grigolob@alma.unibo.it
Roseti
Livia
L
De Franceschi
Luciana
L
Piacentini
Anna
A
Cattini
Luca
L
Manfredini
Massimiliano
M
Faccini
Riccardo
R
Facchini
Andrea
A
D000328:Adult; D053674:Aggrecans; D002356:Cartilage / Q000166:cytology*; D002358:Cartilage, Articular / Q000601:surgery; D002401:Cathepsin B / Q000235:genetics; D019902:Chondrocytes / Q000637:transplantation*; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics; D003094:Collagen / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D051766:Early Growth Response Protein 1; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005260:Female; D006609:High Mobility Group Proteins / Q000235:genetics; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D007150:Immunohistochemistry; D037181:Lectins, C-Type; D008297:Male; D011509:Proteoglycans / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics; D014182:Transplantation, Autologous; D016335:Zinc Fingers / Q000235:genetics
D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D016326:Extracellular Matrix Proteins; D006609:High Mobility Group Proteins; D017874:Immediate-Early Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors; D003094:Collagen; D002401:Cathepsin B