| Summary: | 博士 === 國立陽明大學 === 醫學工程研究所 === 100 === Articular cartilage regeneration is still a challenging issue because of poor spontaneous healing ability, limited cartilage donor site available, fibrosis of the regenerative tissue, and poor integration between native and regenerative cartilages. There are also problems of complicated culture process and increasing carcinogenic risk by using the conventional methods to expand isolated chondrocytes. We therefore propose a study using a novel multiple layer composite scaffold, [GTG/HA-CMC/HA gel/periosteum], to repair osteochondral defect. The GTG (gelatin tricalcium phosphate and glutaraldehyde crosslinked) block was implanted to the inner portion of the defect to repair subchondral bone and also to support regenerated cartilage. In addition, HA(hyaluronic acid) gel was added to provide a 3D matrix containing bio-signal for chondrogenesis of Cambium layer of periosteal membranes. A HA-CMC (hyaluronic acid - carboxymethylcellulose) membrane was placed between these two components as a barrier to prevent vascular invasion before cartilage layer regenerated. The top of this tri-layer scaffold was covered by the periosteal membrane to provide a source of chondrogenic progenitor cells and autologous growth factors.
In this study, we employed an animal model with osteochondral defect sized 4mm in diameter and 5-6mm in depth at medial femoral condyle of rear leg of New Zealand white rabbit. The defects implanted with [GTG/HA-CMC/HA gel/periosteum] multiple-layer composite scaffold act as study group, and the defects implanted with GTG block alone as control group. The regenerated tissue was retrieved from the sacrificed animal 1, 2, 3, 4 and 6 months after operation and studied with aid of histochemical stain and modified Pineda score. There are 25 rabbits enrolled in this study. Regeneration of cartilage in the study group is evidently superior to the control group based on the morphological analyses. The 1st and 2nd months groups revealed regenerated cartilage of zonal arranged characteristic lacunar cells, GAG (glycosaminoglycan) rich ECM (extracellular matrix), and close junction between native and newly-growth cartilages. More mature regenerated cartilage with characteristic structure similar to native cartilage can be found on the specimens of 3rd- and 4th-month groups. The regenerated subchondral bone is fused with native bone without gap. The tide mark could be identified on study group after 4th month. For the control group, the defects were repaired with thin regenerative tissue composed of fibrocartilage without zonal arrangement nor GAG stained ECM although the subchondral bone restored. The modified Pineda scorings of 1st-, 3rd-, 4th-, 6th-month study groups are significantly superior to the control groups. The results also show that the multiple-layer composite scaffold we used could prevent vessel invasion to neo-cartilage and subsequent fibrous change successfully. We also performed experiments by adding TGF-β3 to HA gel. The cartilage layers present over-thickness, fibrous and calcified change because of active inflammatory reaction. The results of quantification of Alcian Blue staining intensity by Image J software are highly correlated to the results reported by pathologist. This digitalized method could be used to improve the objectivity of the Pineda score in future work.
In conclusion, the multiple-layer composite scaffold system we proposed could successfully repair osteochondral defect with neo-cartilage and subchondral bone.
|
|---|
