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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

RT-PCR results of gene expression in AT-MSCs. Premature gene expression in AT-MSCs (A) and specific marker gene expression in AT-MSCs induced by tri-lineage differentiation medium (B) were confirmed.
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Fig4: RT-PCR results of gene expression in AT-MSCs. Premature gene expression in AT-MSCs (A) and specific marker gene expression in AT-MSCs induced by tri-lineage differentiation medium (B) were confirmed.

Mentions: Porcine AT-MSCs adhering to the bottom of the culture dish were spindle-shaped and proliferated well (Figure 2A), reaching over 1 × 106 and 1 × 107 cells at passage 3 and passage 4, respectively. A strong shift in MFI on flow cytometry was detected with antibodies against CD90 and CD105 (Figure 3A, B), while no signals were detected with antibodies against CD34 and CD45 (Figure 3C, D). The genetic markers of OCT-4, SOX-2, KLF-4, C-MYC, and NANOG were all positive (Figure 4A). Following osteogenic induction, AT-MSCs aggregated and contracted to form colonies (Figure 2B), and expressions of specific marker genes, including ALP, OC, and ON, were detected (Figure 4B). These cells also showed appropriate characteristics of the stroma, including staining with alizarin red, indicating the presence of calcium apatite crystals (Figure 2B). Reverse transcription PCR (RT-PCR) of AT-MSCs placed in chondrogenic induction medium revealed the expressions of marker genes, including SOX-9 and AGG (Figure 4B). Histological observation of the cell pellets showed a hyaline cartilage-like structure that was positively stained with alcian blue (Figure 2C). Adipogenic induction of the AT-MSCs resulted in adipocyte-like flattened cells with small lipid vesicles that were positively stained with oil red O (Figure 2D). RT-PCR revealed significant increases in adipogenic marker gene expressions such as AP2 and PPAR-γ2 (Figure 4B).Figure 2


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)

RT-PCR results of gene expression in AT-MSCs. Premature gene expression in AT-MSCs (A) and specific marker gene expression in AT-MSCs induced by tri-lineage differentiation medium (B) were confirmed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: RT-PCR results of gene expression in AT-MSCs. Premature gene expression in AT-MSCs (A) and specific marker gene expression in AT-MSCs induced by tri-lineage differentiation medium (B) were confirmed.
Mentions: Porcine AT-MSCs adhering to the bottom of the culture dish were spindle-shaped and proliferated well (Figure 2A), reaching over 1 × 106 and 1 × 107 cells at passage 3 and passage 4, respectively. A strong shift in MFI on flow cytometry was detected with antibodies against CD90 and CD105 (Figure 3A, B), while no signals were detected with antibodies against CD34 and CD45 (Figure 3C, D). The genetic markers of OCT-4, SOX-2, KLF-4, C-MYC, and NANOG were all positive (Figure 4A). Following osteogenic induction, AT-MSCs aggregated and contracted to form colonies (Figure 2B), and expressions of specific marker genes, including ALP, OC, and ON, were detected (Figure 4B). These cells also showed appropriate characteristics of the stroma, including staining with alizarin red, indicating the presence of calcium apatite crystals (Figure 2B). Reverse transcription PCR (RT-PCR) of AT-MSCs placed in chondrogenic induction medium revealed the expressions of marker genes, including SOX-9 and AGG (Figure 4B). Histological observation of the cell pellets showed a hyaline cartilage-like structure that was positively stained with alcian blue (Figure 2C). Adipogenic induction of the AT-MSCs resulted in adipocyte-like flattened cells with small lipid vesicles that were positively stained with oil red O (Figure 2D). RT-PCR revealed significant increases in adipogenic marker gene expressions such as AP2 and PPAR-γ2 (Figure 4B).Figure 2

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