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Nanohydroxyapatite-reinforced chitosan composite hydrogel for bone tissue repair in vitro and in vivo.

Dhivya S, Saravanan S, Sastry TP, Selvamurugan N - J Nanobiotechnology (2015)

Bottom Line: The potential application of hydrogels as three-dimensional (3D) matrices in tissue engineering has gained attention in recent years because of the superior sensitivity, injectability, and minimal invasive properties of hydrogels.The presence of nHAp in the Zn-CS/nHAp/β-GP hydrogel enhanced swelling, protein adsorption, and exogenous biomineralization.The hydrogel was found to be non-toxic to mesenchymal stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, 603203, Tamil Nadu, India. dhivi.shankar@gmail.com.

ABSTRACT

Background: Bone loss during trauma, surgeries, and tumor resection often results in critical-sized bone defects that need to be filled with substitutionary materials. Complications associated with conventional grafting techniques have led to the development of bioactive tissue-engineered bone scaffolds. The potential application of hydrogels as three-dimensional (3D) matrices in tissue engineering has gained attention in recent years because of the superior sensitivity, injectability, and minimal invasive properties of hydrogels. Improvements in the bioactivity and mechanical strength of hydrogels can be achieved with the addition of ceramics. Based on the features required for bone regeneration, an injectable thermosensitive hydrogel containing zinc-doped chitosan/nanohydroxyapatite/beta-glycerophosphate (Zn-CS/nHAp/β-GP) was prepared and characterized, and the effect of nHAp on the hydrogel was examined.

Methods: Hydrogels (Zn-CS/β-GP, Zn-CS/nHAp/β-GP) were prepared using the sol-gel method. Characterization was carried out by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) as well as swelling, protein adsorption, and exogenous biomineralization studies. Expression of osteoblast marker genes was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analyses. In vivo bone formation was studied using a rat bone defect model system.

Results: The hydrogels exhibited sol-gel transition at 37°C. The presence of nHAp in the Zn-CS/nHAp/β-GP hydrogel enhanced swelling, protein adsorption, and exogenous biomineralization. The hydrogel was found to be non-toxic to mesenchymal stem cells. The addition of nHAp to the hydrogel also enhanced osteoblast differentiation under osteogenic conditions in vitro and accelerated bone formation in vivo as seen from the depositions of apatite and collagen.

Conclusions: The synthesized injectable hydrogel (Zn-CS/nHAp/β-GP) showed its potential toward bone formation at molecular and cellular levels in vitro and in vivo. The current findings demonstrate the importance of adding nHAp to the hydrogel, thereby accelerating potential clinical application toward bone regeneration.

No MeSH data available.


Related in: MedlinePlus

Effect of Zn-CS/nHAp/β-GP hydrogel on bone healing in vivo. a Radiographic images of the rat tibial defects obtained 2 weeks after they were filled with Zn-CS/β-GP hydrogel or Zn-CS/nHAp/β-GP hydrogel. b and c represent hematoxylin and eosin staining and collagen staining of the implants, respectively. Closure of the drill hole, formation of new bone, and deposition of collagen were seen in Zn-CS/nHAp/β-GP-treated animals. Dotted yellow circle indicates the drill hole defect and subsequent closure in hydrogel-treated animals. Purple color-stained areas indicate the formation of bone, and blue-colored areas depict the deposited collagen.
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Fig8: Effect of Zn-CS/nHAp/β-GP hydrogel on bone healing in vivo. a Radiographic images of the rat tibial defects obtained 2 weeks after they were filled with Zn-CS/β-GP hydrogel or Zn-CS/nHAp/β-GP hydrogel. b and c represent hematoxylin and eosin staining and collagen staining of the implants, respectively. Closure of the drill hole, formation of new bone, and deposition of collagen were seen in Zn-CS/nHAp/β-GP-treated animals. Dotted yellow circle indicates the drill hole defect and subsequent closure in hydrogel-treated animals. Purple color-stained areas indicate the formation of bone, and blue-colored areas depict the deposited collagen.

Mentions: We next assessed the bone-healing property of the hydrogels in rats with tibial defects as well as the significance of nHAp in the hydrogels. From radiographic images taken 2 weeks post-surgery, better tissue organization was observed in tibiae treated with Zn-CS/nHAp/β-Gp hydrogels than in tibiae treated with Zn-CS/β-GP hydrogels and in control tibiae (Figure 8a). The radiographs showed that, in the control group, the tibial bone defect was devoid of any implanted hydrogel, resulting in a radiolucent gap with no signs of the drill hole being filled. The presence of nHAp in the Zn-CS/nHAp/β-Gp hydrogel resulted in better wound closure and bone formation, in contrast with the findings for hydrogel without nHAp and the control. The radiographs depicted minimal periosteal reaction and smoothening of cortical bone defect edges in the control and Zn-CS/β-GP-treated groups. The shape of the bone defect changed to an oval morphology, suggesting the initiation of bone healing. Histological analysis involving hematoxylin and eosin (H&E) staining (Figure 8b) and collagen staining (Figure 8c) was performed. Initiation of bone formation was found to be more prominent (purple color) in Zn-CS/nHAp/β-GP hydrogel-treated animals, whereas few areas with purple color were observed in Zn-CS/β-Gp hydrogel-treated animals. No apparent bone formation was observed in control animals (Figure 8b). The presence of nHAp in the hydrogel was a key factor in bone crystal deposition and acted as a nucleating site for bone formation. Collagen is the main component of the ECM involved in the bone repair mechanism. It is clear that collagen deposition (blue-colored deposits) was much more intense in Zn-CS/nHAp/β-GP hydrogel-treated animals than in the control or Zn-CS/β-GP hydrogel-treated animals (Figure 8c). Thus, the addition of nHAp in the Zn-CS/β-GP hydrogel improved bone formation and closure of rat tibial bone defects in vivo.Figure 8


Nanohydroxyapatite-reinforced chitosan composite hydrogel for bone tissue repair in vitro and in vivo.

Dhivya S, Saravanan S, Sastry TP, Selvamurugan N - J Nanobiotechnology (2015)

Effect of Zn-CS/nHAp/β-GP hydrogel on bone healing in vivo. a Radiographic images of the rat tibial defects obtained 2 weeks after they were filled with Zn-CS/β-GP hydrogel or Zn-CS/nHAp/β-GP hydrogel. b and c represent hematoxylin and eosin staining and collagen staining of the implants, respectively. Closure of the drill hole, formation of new bone, and deposition of collagen were seen in Zn-CS/nHAp/β-GP-treated animals. Dotted yellow circle indicates the drill hole defect and subsequent closure in hydrogel-treated animals. Purple color-stained areas indicate the formation of bone, and blue-colored areas depict the deposited collagen.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4464993&req=5

Fig8: Effect of Zn-CS/nHAp/β-GP hydrogel on bone healing in vivo. a Radiographic images of the rat tibial defects obtained 2 weeks after they were filled with Zn-CS/β-GP hydrogel or Zn-CS/nHAp/β-GP hydrogel. b and c represent hematoxylin and eosin staining and collagen staining of the implants, respectively. Closure of the drill hole, formation of new bone, and deposition of collagen were seen in Zn-CS/nHAp/β-GP-treated animals. Dotted yellow circle indicates the drill hole defect and subsequent closure in hydrogel-treated animals. Purple color-stained areas indicate the formation of bone, and blue-colored areas depict the deposited collagen.
Mentions: We next assessed the bone-healing property of the hydrogels in rats with tibial defects as well as the significance of nHAp in the hydrogels. From radiographic images taken 2 weeks post-surgery, better tissue organization was observed in tibiae treated with Zn-CS/nHAp/β-Gp hydrogels than in tibiae treated with Zn-CS/β-GP hydrogels and in control tibiae (Figure 8a). The radiographs showed that, in the control group, the tibial bone defect was devoid of any implanted hydrogel, resulting in a radiolucent gap with no signs of the drill hole being filled. The presence of nHAp in the Zn-CS/nHAp/β-Gp hydrogel resulted in better wound closure and bone formation, in contrast with the findings for hydrogel without nHAp and the control. The radiographs depicted minimal periosteal reaction and smoothening of cortical bone defect edges in the control and Zn-CS/β-GP-treated groups. The shape of the bone defect changed to an oval morphology, suggesting the initiation of bone healing. Histological analysis involving hematoxylin and eosin (H&E) staining (Figure 8b) and collagen staining (Figure 8c) was performed. Initiation of bone formation was found to be more prominent (purple color) in Zn-CS/nHAp/β-GP hydrogel-treated animals, whereas few areas with purple color were observed in Zn-CS/β-Gp hydrogel-treated animals. No apparent bone formation was observed in control animals (Figure 8b). The presence of nHAp in the hydrogel was a key factor in bone crystal deposition and acted as a nucleating site for bone formation. Collagen is the main component of the ECM involved in the bone repair mechanism. It is clear that collagen deposition (blue-colored deposits) was much more intense in Zn-CS/nHAp/β-GP hydrogel-treated animals than in the control or Zn-CS/β-GP hydrogel-treated animals (Figure 8c). Thus, the addition of nHAp in the Zn-CS/β-GP hydrogel improved bone formation and closure of rat tibial bone defects in vivo.Figure 8

Bottom Line: The potential application of hydrogels as three-dimensional (3D) matrices in tissue engineering has gained attention in recent years because of the superior sensitivity, injectability, and minimal invasive properties of hydrogels.The presence of nHAp in the Zn-CS/nHAp/β-GP hydrogel enhanced swelling, protein adsorption, and exogenous biomineralization.The hydrogel was found to be non-toxic to mesenchymal stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, 603203, Tamil Nadu, India. dhivi.shankar@gmail.com.

ABSTRACT

Background: Bone loss during trauma, surgeries, and tumor resection often results in critical-sized bone defects that need to be filled with substitutionary materials. Complications associated with conventional grafting techniques have led to the development of bioactive tissue-engineered bone scaffolds. The potential application of hydrogels as three-dimensional (3D) matrices in tissue engineering has gained attention in recent years because of the superior sensitivity, injectability, and minimal invasive properties of hydrogels. Improvements in the bioactivity and mechanical strength of hydrogels can be achieved with the addition of ceramics. Based on the features required for bone regeneration, an injectable thermosensitive hydrogel containing zinc-doped chitosan/nanohydroxyapatite/beta-glycerophosphate (Zn-CS/nHAp/β-GP) was prepared and characterized, and the effect of nHAp on the hydrogel was examined.

Methods: Hydrogels (Zn-CS/β-GP, Zn-CS/nHAp/β-GP) were prepared using the sol-gel method. Characterization was carried out by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) as well as swelling, protein adsorption, and exogenous biomineralization studies. Expression of osteoblast marker genes was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analyses. In vivo bone formation was studied using a rat bone defect model system.

Results: The hydrogels exhibited sol-gel transition at 37°C. The presence of nHAp in the Zn-CS/nHAp/β-GP hydrogel enhanced swelling, protein adsorption, and exogenous biomineralization. The hydrogel was found to be non-toxic to mesenchymal stem cells. The addition of nHAp to the hydrogel also enhanced osteoblast differentiation under osteogenic conditions in vitro and accelerated bone formation in vivo as seen from the depositions of apatite and collagen.

Conclusions: The synthesized injectable hydrogel (Zn-CS/nHAp/β-GP) showed its potential toward bone formation at molecular and cellular levels in vitro and in vivo. The current findings demonstrate the importance of adding nHAp to the hydrogel, thereby accelerating potential clinical application toward bone regeneration.

No MeSH data available.


Related in: MedlinePlus