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A preliminary study of osteochondral regeneration using a scaffold-free three-dimensional construct of porcine adipose tissue-derived mesenchymal stem cells.

Murata D, Tokunaga S, Tamura T, Kawaguchi H, Miyoshi N, Fujiki M, Nakayama K, Misumi K - J Orthop Surg Res (2015)

Bottom Line: The histopathology of the implants after 6 months revealed active endochondral ossification underneath the plump fibrocartilage in animal no. 1.The histopathology after 12 months in animal no. 2 showed not only that the diminishing fibrocartilage was as thick as the surrounding normal cartilage but also that massive subchondral bone was present.The present results suggest that implantation of a scaffold-free 3D construct of AT-MSCs into an osteochondral defect may induce regeneration of the original structure of the cartilage and subchondral bone over the course of 1 year, although more experimental cases are needed.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Surgery, Department of Veterinary Clinical Science, Joint Faculty of Veterinary Medicine, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima, 890-0065, Japan. daiki_net_offficial@yahoo.co.jp.

ABSTRACT

Background: Osteoarthritis (OA) is a major joint disease in humans and many other animals. Consequently, medical countermeasures for OA have been investigated diligently. This study was designed to examine the regeneration of articular cartilage and subchondral bone using three-dimensional (3D) constructs of adipose tissue-derived mesenchymal stem cells (AT-MSCs).

Methods: AT-MSCs were isolated and expanded until required for genetical and immunological analysis and construct creation. A construct consisting of about 760 spheroids that each contained 5.0 × 10(4) autologous AT-MSCs was implanted into an osteochondral defect (diameter: 4 mm; depth: 6 mm) created in the femoral trochlear groove of two adult microminipigs. After implantation, the defects were monitored by computed tomography every month for 6 months in animal no. 1 and 12 months in animal no. 2.

Results: AT-MSCs were confirmed to express the premature genes and to be positive for CD90 and CD105 and negative for CD34 and CD45. Under specific nutrient conditions, the AT-MSCs differentiated into osteogenic, chondrogenic, and adipogenic lineages, as evidenced by the expressions of related marker genes and the production of appropriate matrix molecules. A radiopaque area emerged from the boundary between the bone and the implant and increased more steadily upward and inward for the implants in both animal no. 1 and animal no. 2. The histopathology of the implants after 6 months revealed active endochondral ossification underneath the plump fibrocartilage in animal no. 1. The histopathology after 12 months in animal no. 2 showed not only that the diminishing fibrocartilage was as thick as the surrounding normal cartilage but also that massive subchondral bone was present.

Conclusions: The present results suggest that implantation of a scaffold-free 3D construct of AT-MSCs into an osteochondral defect may induce regeneration of the original structure of the cartilage and subchondral bone over the course of 1 year, although more experimental cases are needed.

No MeSH data available.


Related in: MedlinePlus

Histopathology of osteochondral defects using Masson’s trichrome, alcian blue, and immunohistochemical staining of type II collagen. In animal no. 1, the articular surface was smooth and fibrocartilage developed on the subchondral bone at the implanted site (A, B, E, F), whereas the surface was irregular and fibrous tissue lay over the subchondral bone at the control site (C, D, G, H). At the implanted site in animal no. 2, the subchondral bone was symmetrically reconstructed and was covered by matrix including hyaline cartilage, which was suggested by the clusters (arrowhead) and columnar clusters (arrow) of cells (I, J, M, N). On the other hand, smooth and continuous surface was restored due to fibrocartilage formation, but subchondral bone was absent in the bottom half of the defect, at the control site in animal no. 2 (K, L, O, P). Black dotted lines indicate the areas of osteochondral defects immediately after the surgery. Masson’s trichrome staining sections (B, D, J, L) were enlarged from red dotted square in the images A, C, I, and K, respectively. The insert images in sections B, D, J, and L were enlarged from white dotted square in images B, D, J, and L, respectively. The bars in the insert images indicate 50 μm.
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Fig7: Histopathology of osteochondral defects using Masson’s trichrome, alcian blue, and immunohistochemical staining of type II collagen. In animal no. 1, the articular surface was smooth and fibrocartilage developed on the subchondral bone at the implanted site (A, B, E, F), whereas the surface was irregular and fibrous tissue lay over the subchondral bone at the control site (C, D, G, H). At the implanted site in animal no. 2, the subchondral bone was symmetrically reconstructed and was covered by matrix including hyaline cartilage, which was suggested by the clusters (arrowhead) and columnar clusters (arrow) of cells (I, J, M, N). On the other hand, smooth and continuous surface was restored due to fibrocartilage formation, but subchondral bone was absent in the bottom half of the defect, at the control site in animal no. 2 (K, L, O, P). Black dotted lines indicate the areas of osteochondral defects immediately after the surgery. Masson’s trichrome staining sections (B, D, J, L) were enlarged from red dotted square in the images A, C, I, and K, respectively. The insert images in sections B, D, J, and L were enlarged from white dotted square in images B, D, J, and L, respectively. The bars in the insert images indicate 50 μm.

Mentions: Histopathological sections of animal no. 1 at 6 months after surgery showed that thickened fibrocartilage had developed over the subchondral bone that was regenerating in the implanted site (Figure 7A, B). The surface of the cartilage was smooth, and the boundary with the surrounding normal cartilage was obscure at the implanted site (Figure 7A). Meanwhile, the surface was collapsed and irregular at the control site (Figure 7C, D). The fibrocartilage showed more intense alcian blue staining and Col-II immunostaining at the implanted site (Figure 7E, F) compared with the control site (Figure 7G, H). In animal no. 2 at 12 months after surgery, partially thickened fibrocartilage was mounted on developed subchondral bone at the implanted site (Figure 7I, J). The surface of the cartilage was smooth, and the boundary with the surrounding normal cartilage was obscure, although small areas of endochondral ossification persisted at the center, and small amounts of AT had differentiated at the bottom part of the site (Figure 7I). Subchondral bone was symmetrically reconstructed in the defect and was covered by a mixed matrix of hyaline cartilage and fibrocartilage, in which clusters and columnar clusters of cells were observed (Figure 7J). In the control site, fibrocartilage had immediately covered the defect, but the subchondral ossification was poor (Figure 7K, L). The hyaline cartilage showed more intense and uniform alcian blue staining and Col-II immunostaining at the implanted site (Figure 7M, N) compared with the control site (Figure 7O, P). The averages of histologic scores for the implanted site were distinctly higher than those for the control site in both animals (Table 4).Figure 7


A preliminary study of osteochondral regeneration using a scaffold-free three-dimensional construct of porcine adipose tissue-derived mesenchymal stem cells.

Murata D, Tokunaga S, Tamura T, Kawaguchi H, Miyoshi N, Fujiki M, Nakayama K, Misumi K - J Orthop Surg Res (2015)

Histopathology of osteochondral defects using Masson’s trichrome, alcian blue, and immunohistochemical staining of type II collagen. In animal no. 1, the articular surface was smooth and fibrocartilage developed on the subchondral bone at the implanted site (A, B, E, F), whereas the surface was irregular and fibrous tissue lay over the subchondral bone at the control site (C, D, G, H). At the implanted site in animal no. 2, the subchondral bone was symmetrically reconstructed and was covered by matrix including hyaline cartilage, which was suggested by the clusters (arrowhead) and columnar clusters (arrow) of cells (I, J, M, N). On the other hand, smooth and continuous surface was restored due to fibrocartilage formation, but subchondral bone was absent in the bottom half of the defect, at the control site in animal no. 2 (K, L, O, P). Black dotted lines indicate the areas of osteochondral defects immediately after the surgery. Masson’s trichrome staining sections (B, D, J, L) were enlarged from red dotted square in the images A, C, I, and K, respectively. The insert images in sections B, D, J, and L were enlarged from white dotted square in images B, D, J, and L, respectively. The bars in the insert images indicate 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389925&req=5

Fig7: Histopathology of osteochondral defects using Masson’s trichrome, alcian blue, and immunohistochemical staining of type II collagen. In animal no. 1, the articular surface was smooth and fibrocartilage developed on the subchondral bone at the implanted site (A, B, E, F), whereas the surface was irregular and fibrous tissue lay over the subchondral bone at the control site (C, D, G, H). At the implanted site in animal no. 2, the subchondral bone was symmetrically reconstructed and was covered by matrix including hyaline cartilage, which was suggested by the clusters (arrowhead) and columnar clusters (arrow) of cells (I, J, M, N). On the other hand, smooth and continuous surface was restored due to fibrocartilage formation, but subchondral bone was absent in the bottom half of the defect, at the control site in animal no. 2 (K, L, O, P). Black dotted lines indicate the areas of osteochondral defects immediately after the surgery. Masson’s trichrome staining sections (B, D, J, L) were enlarged from red dotted square in the images A, C, I, and K, respectively. The insert images in sections B, D, J, and L were enlarged from white dotted square in images B, D, J, and L, respectively. The bars in the insert images indicate 50 μm.
Mentions: Histopathological sections of animal no. 1 at 6 months after surgery showed that thickened fibrocartilage had developed over the subchondral bone that was regenerating in the implanted site (Figure 7A, B). The surface of the cartilage was smooth, and the boundary with the surrounding normal cartilage was obscure at the implanted site (Figure 7A). Meanwhile, the surface was collapsed and irregular at the control site (Figure 7C, D). The fibrocartilage showed more intense alcian blue staining and Col-II immunostaining at the implanted site (Figure 7E, F) compared with the control site (Figure 7G, H). In animal no. 2 at 12 months after surgery, partially thickened fibrocartilage was mounted on developed subchondral bone at the implanted site (Figure 7I, J). The surface of the cartilage was smooth, and the boundary with the surrounding normal cartilage was obscure, although small areas of endochondral ossification persisted at the center, and small amounts of AT had differentiated at the bottom part of the site (Figure 7I). Subchondral bone was symmetrically reconstructed in the defect and was covered by a mixed matrix of hyaline cartilage and fibrocartilage, in which clusters and columnar clusters of cells were observed (Figure 7J). In the control site, fibrocartilage had immediately covered the defect, but the subchondral ossification was poor (Figure 7K, L). The hyaline cartilage showed more intense and uniform alcian blue staining and Col-II immunostaining at the implanted site (Figure 7M, N) compared with the control site (Figure 7O, P). The averages of histologic scores for the implanted site were distinctly higher than those for the control site in both animals (Table 4).Figure 7

Bottom Line: The histopathology of the implants after 6 months revealed active endochondral ossification underneath the plump fibrocartilage in animal no. 1.The histopathology after 12 months in animal no. 2 showed not only that the diminishing fibrocartilage was as thick as the surrounding normal cartilage but also that massive subchondral bone was present.The present results suggest that implantation of a scaffold-free 3D construct of AT-MSCs into an osteochondral defect may induce regeneration of the original structure of the cartilage and subchondral bone over the course of 1 year, although more experimental cases are needed.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Surgery, Department of Veterinary Clinical Science, Joint Faculty of Veterinary Medicine, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima, 890-0065, Japan. daiki_net_offficial@yahoo.co.jp.

ABSTRACT

Background: Osteoarthritis (OA) is a major joint disease in humans and many other animals. Consequently, medical countermeasures for OA have been investigated diligently. This study was designed to examine the regeneration of articular cartilage and subchondral bone using three-dimensional (3D) constructs of adipose tissue-derived mesenchymal stem cells (AT-MSCs).

Methods: AT-MSCs were isolated and expanded until required for genetical and immunological analysis and construct creation. A construct consisting of about 760 spheroids that each contained 5.0 × 10(4) autologous AT-MSCs was implanted into an osteochondral defect (diameter: 4 mm; depth: 6 mm) created in the femoral trochlear groove of two adult microminipigs. After implantation, the defects were monitored by computed tomography every month for 6 months in animal no. 1 and 12 months in animal no. 2.

Results: AT-MSCs were confirmed to express the premature genes and to be positive for CD90 and CD105 and negative for CD34 and CD45. Under specific nutrient conditions, the AT-MSCs differentiated into osteogenic, chondrogenic, and adipogenic lineages, as evidenced by the expressions of related marker genes and the production of appropriate matrix molecules. A radiopaque area emerged from the boundary between the bone and the implant and increased more steadily upward and inward for the implants in both animal no. 1 and animal no. 2. The histopathology of the implants after 6 months revealed active endochondral ossification underneath the plump fibrocartilage in animal no. 1. The histopathology after 12 months in animal no. 2 showed not only that the diminishing fibrocartilage was as thick as the surrounding normal cartilage but also that massive subchondral bone was present.

Conclusions: The present results suggest that implantation of a scaffold-free 3D construct of AT-MSCs into an osteochondral defect may induce regeneration of the original structure of the cartilage and subchondral bone over the course of 1 year, although more experimental cases are needed.

No MeSH data available.


Related in: MedlinePlus