Fig 1.
The young adult axolotl, Ambystoma mexicaum.
Left, wild-type animal with dark skin pigmentation. Right, the white mutant, which lacks skin melanophores.
Table 1.
Growth factors implicated by bioinformatics analysis in cartilage and bone regeneration, and in fracture repair.
Table 2.
Growth factor combinations and concentrations tested for their ability to promote cartilage regeneration across 50% defects in the axolotl fibula.
Table 3.
Regeneration of cartilage in the absence of intervention across defects of 10%, 20%, 40% and 50% in the axolotl fibula.
Fig 2.
10% and 20% defects imaged by micro-CT at two months post-operation.
The tibia is to the top and the fibula at the bottom. Lines indicate the boundaries of the regenerated bone. Note that these boundaries represent the bulk of the regenerated bone, which extends beyond the defect region per se.
Fig 3.
Whole mounts of control 10% (A, B, C) and 20% defects (D, E, F) stained with methylene blue (A, D) or methylene blue/alizarin red (B, E), and H & E-stained sections(C, F), three months post-operation.
Tibia is on the left and fibula on the right in all photos. Both cartilage and bone (arrows) have regenerated across the defects. Bars in (C, D) equal 400 μm.
Fig 4.
Control 40% (A, C) and 50% (B, D) defects three months post-operation.
(A, B), whole mounts stained with methylene blue/alizarin red. C, D, H & E-stained sections. No regeneration has taken place. In the sections, muscle and connective tissue have filled the defect space (arrows). T = tibia. Bars = 400 μm.
Fig 5.
Bone volume fraction of regenerated bone in 10%- 50% defects at two months post-operation.
The 20% defect is the same one illustrated in Fig 2. Top, red dashed lines show the cross-section level in the center of the 10% and 20% defects (A, B) and the 40% and 50% defects (C, D) where the measurements were taken. Both 10% and 20% defects regenerated a high volume fraction of bone, whereas there was much less regeneration in the 40% defects, and minimal regeneration in the 50% defects, which only regenerated at the cut ends of the fibula.
Fig 6.
Use of two fluorochromes to measure bone regeneration in 10% and 50% segment defects after 3 weeks of regeneration.
1% calcein was injected on the day of surgery at a dose of 15ug/mg body weight. 2% alizarine complexone red was injected 1 week post-surgery at a dose of 30ug/mg body weight. Scale bar = 50 μM. Samples (six for each group) were harvested 3 weeks post-surgery. (A-1, A-2, A-3) is a 10% defect; (B-1,B-2,B-3) is a 50% defect. (A-1, B-1), calcein green fluorescence; (A2, B2), alizarine complexone red fluorescence; (A-3, B-3), red and green color merged. In the merged image of the 10% defect (A-3), the red color extends beyond the green, indicating that regeneration is taking place. By contrast, red and green show no separation in the merged image of the 50% defect (B-3), indicating lack of regeneration. The extent of regeneration was obtained by measuring the length of red extending beyond green on the anterior, middle and posterior points of the anterior-posterior axis of the fibula and averaging the three measurements (bar graph). The difference between the 10% and 20% defects was significant at p = 0.05 (Unpaired T-test). Scale bar = 50 μm.
Fig 7.
Result of implanting 8-braid SIS scaffold soaked in 0.8 aPBS.
Left, 8-braid scaffold prior to hydration. Middle and right, 50% defect at one and two months, respectively, after embedding SIS scaffold alone. No cartilage has regenerated. In the one-month specimen, the implanted scaffold is still visible within the gap (arrow). At two months, the scaffold is largely degraded, and connective tissue and muscle has regenerated into the gap (arrow). M = muscle. Scale bar = 400 μm.
Table 4.
Number of limbs in which growth factors and limb tissue extract induced partial or extensive regeneration across 50% defects in the axolotl fibula at three months post-operation.
Fig 8.
A 50% defect treated with BMP4/VEGF/HGF/EGF/FGF2/PDGF-AA/TGFβ-3, three months post-implant.
A small amount of cartilage (arrow) regenerated from the proximal end of the fibula. The distal end of the fibula (DF) was severely angled with respect to the proximal end. A small amount of cartilage appears to have regenerated transversely on the distal fibula stump (asterisk). T = tibia. Scale bar = 400 μm.
Fig 9.
A 50% defect treated with BMP4/VEGF, three months post-operation.
An irregular tongue of cartilage with periosteal bone collar (arrow) has regenerated from the distal end of the fibula. Remnants of the SIS scaffold can be seen within the defect, along with muscle and connective tissue (arrowhead). T = tibia. Scale bar = 400 μm.
Fig 10.
Left, MB-stained whole mount of 50% defect treated with BMP4/HGF, 3 months post-operation.
A short cone of cartilage (arrow) regenerated from the proximal end of the fibula. No regeneration took place from the distal end. Right, MB-stained whole mount of 50% defect treated with BMP4/HGF in which short cones of cartilage regenerated from both the proximal and distal ends of the fibula (arrows). Both specimens illustrate a common phenomenon encountered after removal of 50% of the bone, namely that the remaining distal and/or proximal bone segments regress to create closer to a 70% gap.
Fig 11.
A 50% defect treated with BMP4/HGF, three months post-operation.
An irregular bar of cartilage (asterisk) was induced along the proximodistal axis of the tibia (T). Horizontal lines indicate the boundaries of the gap in the fibula. Scale bar = 400 μm.
Fig 12.
A 50% defect three months after treatment with BMP4/HGF, in which cartilage (asterisk) regenerated proximally from the distal end of the fibula (F) across half the defect.
A thin shell of periosteal bone (arrow) covers the regenerated cartilage. M = muscle. Scale bar = 400 μm.
Fig 13.
A 50% defect three months after treatment with BMP-4/HGF.
Cartilage (arrow) has regenerated across the defect space. Proximal is to the right; distal to the left. Vertical lines indicate approximate proximodistal boundaries of the defect space. T = tibia. Scale bar = 400 μm.
Fig 14.
A 50% defect three months after treatment with tissue extract.
Cartilage and bone regenerated over nearly the complete length of the defect (arrow). Bone marrow (BM) has formed in the middle of the regenerated bone. T = tibia. Scale bar = 400 μm.
Fig 15.
(A) Methylene blue-stained whole mount of a 50% defect three months after treatment with tissue extract.
The gap has been completely bridged by an irregular mass of cartilage, which appears to have regenerated primarily from the distal end of the fibula, with only a sliver regenerating from the proximal end (arrow). A cartilage bridge (asterisk), most likely derived from the tibial periosteum, connects the regenerating fibula to the tibia (T). (B) Methylene blue/alizarin red-stained whole mount of a 50% defect three months after treatment with tissue extract. The ends of the fibula were angled with respect to one another so that regeneration from the proximal and distal ends produced a V shape. A supernumerary foot (asterisk) regenerated perpendicular to the fibula. The star indicates the normal foot. Distal is toward the top; proximal is toward the bottom. F = femur; T = tibia; Fb = fibula.
Fig 16.
Micro-CT scan of specimen in Fig 15B.
The arrow points to the regenerated fibula. S = supernumerary foot skeletal elements; P = skeletal elements of the primary foot. F = femur; T = tibia. R = remnant of distal cut end of the fibula, which has regressed extensively.
Fig 17.
Release kinetics of BMP4 from 8-braid SIS scaffold.
Three samples were measured per time point. Bars = SD.