R.S. Cosden-Decker, M.M. Bickett, C. Lattermann, J.N. MacLeod

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Presentation transcript:

Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders R.S. Cosden-Decker, M.M. Bickett, C. Lattermann, J.N. MacLeod Osteoarthritis and Cartilage Volume 20, Issue 11, Pages 1347-1356 (November 2012) DOI: 10.1016/j.joca.2012.07.002 Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Tibial defect model in the axolotl salamander. (A) Tibial defect location and (B) hind limb anatomy schematic, adapted from Ref. 36. (C) Surgical exposure of the tibia diaphysis prior to gap fracture creation. (D) The critical size tibial defects were made by removing a 4 mm length of tibial diaphysis. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Diarthrodial joint anatomy in axolotls greater than 12 months of age. Whole mount Aliziran Red and Alician Blue double staining of cartilage and bone structures in the (A) proximal femur, (D) radiohumoral, (G) femorotibial, and (J) carpal-metacarpal-phalangeal structures. Histological analyses reveal that cavitation occurs in the more proximal joints including femoroacetabular (B, C) and radiohumoral (E, F) articulations, while an interzone-like tissue is retained in more distal joints including the femorotibial (H, I), carpal (K), and interphalangeal (L) articulations. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Changes in cell morphology of tissue within the axolotl femorotibial joint from 2 to 24 months of age. Distal femoral cartilage (A–E) and interzone-like tissue of axolotl salamanders at 2, 4, 7, 12 and 24 months of age (F–J). Scale bar lengths are indicated. EC, epiphyseal/articular cartilage. MC, metaphyseal cartilage. MC-M, morphologically distinct metaphyseal cartilage zone adjacent to the medullary cavity. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Changes in cellularity of axolotl epiphyseal/articular cartilage in the distal femur and interzone-like tissue in the femorotibial joint from 2 to 24 months of age (mean ± SD). *Denotes difference in cellularity from groups after 7 months of age (P < 0.05). Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Expression of joint markers in mature axolotls. Type I collagen (A, F), type II collagen (B, G), Aggrecan (C, H), Proteoglycans (D, I), BOC (E) and GDF5 (J) in the distal femoral cartilage and interzone-like tissue of axolotl salamanders at greater than 12 months of age. Type I collagen, type II collagen, aggrecan, BOC, and GDF5 were detected by immunohistochemistry. Proteoglycan was visualized by Safranin-O staining. Images A–E are shown at 4× magnification, while images F–J are shown at 20× magnification. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Tibial fracture repair in axolotl salamanders. Whole mount bone (Aliziran Red) and cartilage (Alcian Blue) staining of (A–C) transverse bone fractures, (D–F) CSD, and (G–I) CSD with interzone tissue transplant at 15 days (A, D, G), 6 weeks (B, E, H), and 7 months (C, F, I) after surgery. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Repair of critical size tibial defects with interzone-like tissue transplants at 7 months post-surgery. (A) Whole mount Alcian Blue and Alizarin Red staining and (B) H&E staining reveal formation of an accessory joint (box) including interzone tissue (arrowheads) within the tibial defect space. (C) Safranin-O stained image demonstrates metaphyseal cartilage (MC), epiphyseal/articular cartilage (EC) and interzone tissue (IZ, arrows) within the accessory joint. (D) All tissues within the accessory joint displayed positive immunostaining for GFP confirming their development from the original interzone tissue transplant. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Supplemental Fig. 1 Immunohistochemistry controls. Positive and negative controls for immunohistochemical localization of aggrecan, BOC, type I collagen, type II collagen and GDF5 in axolotl joint tissues. Equine and murine samples were used as positive controls. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Supplemental Fig. 2 Spontaneous repair of a tibial gap fracture after fibula bowing Bone (Alizarin Red) and cartilage (Alcian Blue) staining of a normal axolotl hind limb (A). Bowing of the fibula (arrows) was observed in a few individuals after a 4 mm surgical resection of tibial diaphysis at 6 weeks (B) and 7 months (C) post-surgery which shortened the tibial gap and enabled spontaneous repair. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Supplemental Fig. 3 Safranin-O/Fast Green staining of critical size bone defects with muscle (A, C) or skin (B, D) transplants at 7 months after surgery. Fracture ends became rounded with evidence of cartilaginous cap formation, but the critical size defects failed to heal in the absence of interzone tissue transplants. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Supplemental Table I Axolotl tibial fracture experimental samples. Biological replicates, post-surgical collection time, and experimental processing of transverse fractures, critical size bone defects (CSD), CSD with interzone-like tissue transplants, CSD with muscle transplants and CSD with skin transplants. Osteoarthritis and Cartilage 2012 20, 1347-1356DOI: (10.1016/j.joca.2012.07.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions