Periodic Reporting for period 1 - PONS (Fabrication of Polysaccharides-Protein complex Nano-Hybrid Scaffold as regenerative biomaterial for bone tissue engineering)
Reporting period: 2019-04-01 to 2021-03-31
The Pol-PrO complex reinforced LDH-BCM Nanohybrid Scaffold (PONS) was developed as regenerative biomaterial in bone tissue engineering. The 3D nano hybrid scaffold was developed by optimizing the concentration and the ratio of blending of the Pol-Pro complex. The nano hybrid Pol-Pro complex scaffold was fabricated by incorporating layered double hydroxide coated with rutin as biological carrier molecule to enhance the bone regeneration using freeze drying technology. The nano hybrid scaffold were physio-chemically and biologically evaluated for application in bone tissue engineering. Thus this proposal aim was attained by fabricated an organic-inorganic complex nanohybrid scaffold as a bone implant for applications in tissue.
- The project has achieved most of its objectives and milestones for the period, with relatively minor deviations.
O1.Synthesis and characterization of BMP2 conjugated LDH nanohybrid as the functional biological career molecule
O2.Fabrication and evaluation of the LDH nanohybrid reinforced Polysaccharide-Protein (Pol-Pro) complex scaffold
O3.Evaluation of the fabricated LDH nanohybrid Pol-Pro complex hybrid scaffold using in vitro, in situ and in vivo studies using mice as the animal model
Objective 1, 2 and 3 was complete successfully as given below. However, part of the objective 3 (WP4) was unable to complete due to the COVID 19 situation.
WP1: Synthesis, Characterization and conjugation of LDH nano-hybrid with BCM :
The two different combination of LDH was prepared using aqueous mixed salt solution (Zn-Fe) by co-precipitation method. The prepared Zn-Fe LDH was characterized and coated with biologically active carrier molecule (BCM) to enhance the osteoblast adhesion and regeneration of bone. For this we have selected Rutin as the BCM and coated over the prepared Zn-Fe LDH and then incorporated into the Pol-Pro hybrid complex scaffold n WP 2.1.
WP2: Design and development of nanohybrid reinforced Pol-Pro complex scaffolds
WP2.1. Fabrication of LDH nano-hybrid reinforced Pol-Pro complex scaffold :
The 3D hierarchical hybrid scaffold was fabricated with different weight percentage of Polysaccharide (Bacterial Cellulose) and Protein (Collagen) and were blended uniformly and poured into pre-designed moulds. The Pol-Pro complex mixture were frozen step by step at different temperature from -4°C, -20°C, -40°C and -80°C at constant time intervals followed by freeze drying for 24 h to obtain three-dimensional (3D) Pol-Pro complex scaffolds. The material with best porosity was chosen to reinforce LDH into the Pol-Pro complex scaffold and similarly, the LDH reinforced Pol-Pro complex scaffold was fabricated. All fabricated scaffold were sterilized and stored at 4°C until further use.
WP2.2. Characterization of the fabricated LDH reinforce Pol-Pro complex scaffold
The chemical and conformational changes of the fabricated scaffold were investigated using Fourier transform infrared (FTIR) measurements, to determine the functional groups. The surface morphology of the scaffolds will be visualized using SEM, and µCT analysis. The scaffolds were also evaluated for the bioactivity or mineralization assay in stimulated body fluid (SBF). The in vitro biodegradation, porosity and specific surface area of the scaffold were evaluated to ensure the oxygen permeability, metabolic exchange, cell seeding efficiency. Compression test.
WP3: Evaluation of in situ and in vitro efficacy of LDH nano-hybrid reinforced Pol-Pro complex scaffolds:
The in vitro evaluation of the fabricated nano-hybrid scaffold was done with bone cell line and we have got good biocompatibility of the scaffold. Along with biocompatibility, the live dead staining of the cells were done.
WP4: Evaluation of the in vivo efficacy of LDH reinforced Pol-Pro complex hybrid 3D scaffolds using mice as the animal model:
As a proof of the biocompatibility, further, in vivo studies on the developed scaffold was proposed to do. But due do the COVID 19 situation I am not unable to procced with that evaluation.
- The project has achieved most of its objectives and milestones for the period, with relatively minor deviations.
O1.Synthesis and characterization of BMP2 conjugated LDH nanohybrid as the functional biological career molecule
O2.Fabrication and evaluation of the LDH nanohybrid reinforced Polysaccharide-Protein (Pol-Pro) complex scaffold
O3.Evaluation of the fabricated LDH nanohybrid Pol-Pro complex hybrid scaffold using in vitro, in situ and in vivo studies using mice as the animal model
Objective 1, 2 and 3 was complete successfully as given below. However, part of the objective 3 (WP4) was unable to complete due to the COVID 19 situation.
WP1: Synthesis, Characterization and conjugation of LDH nano-hybrid with BCM :
The two different combination of LDH was prepared using aqueous mixed salt solution (Zn-Fe) by co-precipitation method. The prepared Zn-Fe LDH was characterized and coated with biologically active carrier molecule (BCM) to enhance the osteoblast adhesion and regeneration of bone. For this we have selected Rutin as the BCM and coated over the prepared Zn-Fe LDH and then incorporated into the Pol-Pro hybrid complex scaffold n WP 2.1.
WP2: Design and development of nanohybrid reinforced Pol-Pro complex scaffolds
WP2.1. Fabrication of LDH nano-hybrid reinforced Pol-Pro complex scaffold :
The 3D hierarchical hybrid scaffold was fabricated with different weight percentage of Polysaccharide (Bacterial Cellulose) and Protein (Collagen) and were blended uniformly and poured into pre-designed moulds. The Pol-Pro complex mixture were frozen step by step at different temperature from -4°C, -20°C, -40°C and -80°C at constant time intervals followed by freeze drying for 24 h to obtain three-dimensional (3D) Pol-Pro complex scaffolds. The material with best porosity was chosen to reinforce LDH into the Pol-Pro complex scaffold and similarly, the LDH reinforced Pol-Pro complex scaffold was fabricated. All fabricated scaffold were sterilized and stored at 4°C until further use.
WP2.2. Characterization of the fabricated LDH reinforce Pol-Pro complex scaffold
The chemical and conformational changes of the fabricated scaffold were investigated using Fourier transform infrared (FTIR) measurements, to determine the functional groups. The surface morphology of the scaffolds will be visualized using SEM, and µCT analysis. The scaffolds were also evaluated for the bioactivity or mineralization assay in stimulated body fluid (SBF). The in vitro biodegradation, porosity and specific surface area of the scaffold were evaluated to ensure the oxygen permeability, metabolic exchange, cell seeding efficiency. Compression test.
WP3: Evaluation of in situ and in vitro efficacy of LDH nano-hybrid reinforced Pol-Pro complex scaffolds:
The in vitro evaluation of the fabricated nano-hybrid scaffold was done with bone cell line and we have got good biocompatibility of the scaffold. Along with biocompatibility, the live dead staining of the cells were done.
WP4: Evaluation of the in vivo efficacy of LDH reinforced Pol-Pro complex hybrid 3D scaffolds using mice as the animal model:
As a proof of the biocompatibility, further, in vivo studies on the developed scaffold was proposed to do. But due do the COVID 19 situation I am not unable to procced with that evaluation.
The research goal of fabricating a 3D nano hybrid scaffold with polysaccharide and protein complex was attained successfully. The Physiochemical and biological evaluation of the fabricated scaffold was performed. The bidirectional transfer of knowledge from researcher to host and host to researcher was acquired with Prof. Fardim group. researcher also attained transfer of knowledge. The researcher has training on the operating procedure and optimization of various polymers into beads using the vibrational, aerodynamic and jet cut technology using NISCO devices. The COVID 19 was the major problem, due to that in vivo part of the fabricated scaffold was not completed.
The projects has enhanced the future career prospects and added as a milestone in the curriculum vitae to good opportunity. Currently I am applying for post doctorate research position in European countries. This work will have a chance of to create new market opportunities, strengthen competitiveness and growth of companies by developing bone implant as product. However, the final WP4 of the project was not completed to attain this goal. Moreover, the fabricated bone implant and the characterization up to in vitro studies will be publication in international journal with good impact factor.