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Gene-Activated Matrix Comprised of Atelocollagen and Plasmid DNA Encoding BMP4 or Runx2 Promotes Rat Cranial Bone Augmentation.

Umebayashi M, Sumita Y, Kawai Y, Watanabe S, Asahina I - Biores Open Access (2015)

Bottom Line: Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture.Then, GAMs were lyophilized and transplanted to onlay placement on the cranium.At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Regenerative Oral Surgery, Unit of Translational Medicine, Graduate School of Biomedical Sciences, Nagasaki University , Nagasaki, Japan .

ABSTRACT
To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though its potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any transfection reagents or virus vectors. In this study, to facilitate the clinical setting of this strategy, particularly aimed at atrophic bone repair, we simply investigated whether manufactured gene-activated matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, or 1 mg of AcGFP plasmid vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine atelocollagen and β-tricalcium phosphate granules. Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 1 mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1 mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1 mg of pBMP4 or pRunx2 at 8 weeks as well. Thus, atelocollagen-based GAM reliably could form the engineered bone even for the vertical augmentation when containing a certain amount of plasmid vectors encoding osteogenic proteins. This study supports facilitating the clinical application of GAM for bone engineering.

No MeSH data available.


Related in: MedlinePlus

Histological observation at 8 weeks of GAM transplantation to onlay placement. (A–C) Representative images of HE staining of specimens in GFP, BMP4, and Runx2 groups, respectively. New bone area was markedly augmented in specimens of BMP4 (B) and Runx2 (C) groups while small amounts of new bone detectable in the GFP group (A). Scale bar is 50 μm. (D) Representative images of osteocalcin immunostaining in the Runx2 group. Scale bar in 50 μm. (E–I) The black box areas in (A–D) are shown in higher magnification. Mature bone tissues, which included osteocytes, were observed in BMP4 (F) and Runx2 (G) groups, while small amounts of new bone formation were recognized in close proximity to β-TCP granules in the GFP group (E). Osteocalcin-positive cells were seen at the surface of new bone tissues (H) and not recognized in control sections treated with preimmune serum (negative control) (I). Scale bar is 10 μm. Yellow dotted line indicates boundary of the host and newly formed bone; black arrow, area of augmented bone; asterisk, β-TCP granules; and black arrow head, replacement to bone at the surface of absorbed β-TCP granules.
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f4: Histological observation at 8 weeks of GAM transplantation to onlay placement. (A–C) Representative images of HE staining of specimens in GFP, BMP4, and Runx2 groups, respectively. New bone area was markedly augmented in specimens of BMP4 (B) and Runx2 (C) groups while small amounts of new bone detectable in the GFP group (A). Scale bar is 50 μm. (D) Representative images of osteocalcin immunostaining in the Runx2 group. Scale bar in 50 μm. (E–I) The black box areas in (A–D) are shown in higher magnification. Mature bone tissues, which included osteocytes, were observed in BMP4 (F) and Runx2 (G) groups, while small amounts of new bone formation were recognized in close proximity to β-TCP granules in the GFP group (E). Osteocalcin-positive cells were seen at the surface of new bone tissues (H) and not recognized in control sections treated with preimmune serum (negative control) (I). Scale bar is 10 μm. Yellow dotted line indicates boundary of the host and newly formed bone; black arrow, area of augmented bone; asterisk, β-TCP granules; and black arrow head, replacement to bone at the surface of absorbed β-TCP granules.

Mentions: The histology of the rat cranium (onlay placement) at 8 weeks postoperatively is shown in Figure 4. At this stage, only small amounts of new bone were detectable in the GFP group containing 1 mg pDNAs (Fig. 4A) and GAMs containing 0.02 or 0.1 mg pDNAs (data not shown). However, when GAMs harboring 1 mg pDNAs were transplanted, we found that new bone area was markedly augmented in specimens of BMP4 and Runx2 groups compared with that in the same groups at 4 weeks (Fig. 4B,C). Absorption of β-TCP granules surrounded by new bone was progressing further, and augmented bone seemed to be mature. Mature bone tissues, which included osteocytes, were observed on the magnified micrographs in BMP4 and Runx2 groups (Fig. 4F,G). In contrast, small amounts of new bone formation were recognized in close proximity to TCP granules in the GFP group (Fig. 3E). Staining areas of osteocalcin were detected obviously in osteoblastic cells and the surface of new bone tissues in BMP4 and Runx2 groups (Fig. 4D,H). Control sections treated with preimmune serum exhibited no reactivity, indicating that the staining was specific (Fig. 4I).


Gene-Activated Matrix Comprised of Atelocollagen and Plasmid DNA Encoding BMP4 or Runx2 Promotes Rat Cranial Bone Augmentation.

Umebayashi M, Sumita Y, Kawai Y, Watanabe S, Asahina I - Biores Open Access (2015)

Histological observation at 8 weeks of GAM transplantation to onlay placement. (A–C) Representative images of HE staining of specimens in GFP, BMP4, and Runx2 groups, respectively. New bone area was markedly augmented in specimens of BMP4 (B) and Runx2 (C) groups while small amounts of new bone detectable in the GFP group (A). Scale bar is 50 μm. (D) Representative images of osteocalcin immunostaining in the Runx2 group. Scale bar in 50 μm. (E–I) The black box areas in (A–D) are shown in higher magnification. Mature bone tissues, which included osteocytes, were observed in BMP4 (F) and Runx2 (G) groups, while small amounts of new bone formation were recognized in close proximity to β-TCP granules in the GFP group (E). Osteocalcin-positive cells were seen at the surface of new bone tissues (H) and not recognized in control sections treated with preimmune serum (negative control) (I). Scale bar is 10 μm. Yellow dotted line indicates boundary of the host and newly formed bone; black arrow, area of augmented bone; asterisk, β-TCP granules; and black arrow head, replacement to bone at the surface of absorbed β-TCP granules.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Histological observation at 8 weeks of GAM transplantation to onlay placement. (A–C) Representative images of HE staining of specimens in GFP, BMP4, and Runx2 groups, respectively. New bone area was markedly augmented in specimens of BMP4 (B) and Runx2 (C) groups while small amounts of new bone detectable in the GFP group (A). Scale bar is 50 μm. (D) Representative images of osteocalcin immunostaining in the Runx2 group. Scale bar in 50 μm. (E–I) The black box areas in (A–D) are shown in higher magnification. Mature bone tissues, which included osteocytes, were observed in BMP4 (F) and Runx2 (G) groups, while small amounts of new bone formation were recognized in close proximity to β-TCP granules in the GFP group (E). Osteocalcin-positive cells were seen at the surface of new bone tissues (H) and not recognized in control sections treated with preimmune serum (negative control) (I). Scale bar is 10 μm. Yellow dotted line indicates boundary of the host and newly formed bone; black arrow, area of augmented bone; asterisk, β-TCP granules; and black arrow head, replacement to bone at the surface of absorbed β-TCP granules.
Mentions: The histology of the rat cranium (onlay placement) at 8 weeks postoperatively is shown in Figure 4. At this stage, only small amounts of new bone were detectable in the GFP group containing 1 mg pDNAs (Fig. 4A) and GAMs containing 0.02 or 0.1 mg pDNAs (data not shown). However, when GAMs harboring 1 mg pDNAs were transplanted, we found that new bone area was markedly augmented in specimens of BMP4 and Runx2 groups compared with that in the same groups at 4 weeks (Fig. 4B,C). Absorption of β-TCP granules surrounded by new bone was progressing further, and augmented bone seemed to be mature. Mature bone tissues, which included osteocytes, were observed on the magnified micrographs in BMP4 and Runx2 groups (Fig. 4F,G). In contrast, small amounts of new bone formation were recognized in close proximity to TCP granules in the GFP group (Fig. 3E). Staining areas of osteocalcin were detected obviously in osteoblastic cells and the surface of new bone tissues in BMP4 and Runx2 groups (Fig. 4D,H). Control sections treated with preimmune serum exhibited no reactivity, indicating that the staining was specific (Fig. 4I).

Bottom Line: Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture.Then, GAMs were lyophilized and transplanted to onlay placement on the cranium.At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Regenerative Oral Surgery, Unit of Translational Medicine, Graduate School of Biomedical Sciences, Nagasaki University , Nagasaki, Japan .

ABSTRACT
To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though its potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any transfection reagents or virus vectors. In this study, to facilitate the clinical setting of this strategy, particularly aimed at atrophic bone repair, we simply investigated whether manufactured gene-activated matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, or 1 mg of AcGFP plasmid vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine atelocollagen and β-tricalcium phosphate granules. Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 1 mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1 mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1 mg of pBMP4 or pRunx2 at 8 weeks as well. Thus, atelocollagen-based GAM reliably could form the engineered bone even for the vertical augmentation when containing a certain amount of plasmid vectors encoding osteogenic proteins. This study supports facilitating the clinical application of GAM for bone engineering.

No MeSH data available.


Related in: MedlinePlus