Scientists at Nanyang Technological University, Singapore (NTU Singapore) have developed a new biomaterial made entirely from bullfrog skin and discarded fish scales that could aid in bone repair.
The porous biomaterial, which contains the same compounds that are predominant in bone, acts as a scaffold for bone-forming cells to adhere and multiply, leading to the formation of new bone.
Through laboratory experiments, the team at NTU Singapore found that human bone cells seeded on the biomaterial scaffold successfully attached and began to multiply – a sign of growth. They also found that the risk of the biomaterial triggering an inflammatory response is low.
Such scaffolding could be used to aid in the regeneration of bone tissue lost due to disease or injury, such as jaw defects due to trauma or cancer surgery. It could also promote bone growth around surgical implants such as dental implants.
Scientists believe the biomaterial is a promising alternative to the current standard practice of using a patient’s own tissue, which requires additional surgery for bone extraction. At the same time, the production of this biomaterial is tackling the problem of aquaculture waste, said assistant professor Dalton Tay of the NTU School of Materials Science and Engineering (MSE), who led the multidisciplinary study.
More than 20 million tonnes of fishery by-products, such as fins, scales and skins, are discarded each year. In Singapore, the combined annual consumption of frog meat and fish is estimated to be around 100 million kilograms, making bullfrog skin and fish scales two of Singapore’s largest secondary streams of aquaculture waste.
Professor Asst Dalton Tay said, âWe took the ‘waste to resource’ approach in our study and turned waste into a high value material with biomedical applications, closing the waste loop in the process. Our lab studies have shown that the biomaterial we engineered could be a promising option that aids in bone repair. The potential of this biomaterial is very wide, ranging from the repair of bone defects due to injury or aging, to dental applications for aesthetics. Our research is based on all of NTU’s work in this field. sustainability and is in line with Singapore’s circular economy approach towards a zero waste nation. “
Professor Matthew Hu Xiao, co-author of the study and director of the Center for Environmental Chemistry and Materials, Nanyang Water and Environment Research Institute (NEWRI), added, âThese waste streams can also be converted to chemicals and green materials for environmental sanitation and in a timely manner. treatment can reduce sewage contamination. “
Associate Clinical Professor Goh Bee Tin, Director of Research at the Singapore National Dental Center, who was not involved in the study, said, âThe Singapore National Dental Center is excited about the use of the skin. bullfrog as a natural biomaterial for tissue regeneration. We see many potential dental applications ranging from regeneration of gum tissue in periodontal disease, to bone for dental implant placement, to jaw bone after tumor surgery. the patients.”
The research results were published online in Materials Science and Engineering C in April and will be published in volume 126 of the journal in July.
The research team has filed for patents for the wound healing and bone tissue engineering applications of the biomaterial. The team is now further evaluating the safety and long-term efficacy of the biomaterial as dental products under a grant from the China-Singapore Joint International Research Institute and aims to bring the technology pipeline closer to recovery of marketing waste.
Turning waste into treasure
With the combined annual consumption of frog meat and fish in Singapore estimated at around 100 million kilograms, bullfrog skin and fish scales are two of Singapore’s largest aquaculture waste secondary streams. Fishing waste used by the NTU team was collected at Khai Seng Fish Farm and Jurong Frog Farm.
To make the biomaterial, the team first extracted type 1 tropocollagen (many molecules of which form collagen fibers) from the discarded skins of the American bull frog, which is grown locally and imported in large numbers to Singapore for consumption. ; and hydroxyapatite (a compound of calcium phosphate) from the scales of snakehead fish, commonly known as Toman fish.
Collagen and hydroxyapatite (HA) are two predominant components found in bone, thus giving the biomaterial a bone-like structure, composition, and ability to promote cell attachment. These two components also make the biomaterial resistant.
Scientists removed all the impurities from the bullfrog skin and then mixed it together to form a thick collagen paste which is diluted with water. Collagen was then extracted from this mixture. “With this approach, we were able to obtain the highest ever reported yield of collagen of around 70% from frog skin, thus making this approach commercially viable,” said Professor Asst Tay, also of the NTU School of Biological Sciences (SBS).
HA was harvested from discarded fish scales by calcination – a purification process that requires high heat – to remove organic matter, and then air dried.
The biomaterial was synthesized by adding HA powder to the extracted collagen, then cast into a mold to produce a 3D porous scaffold. This whole process took less than two weeks and the team believes it can be both shortened and extended.
Proof of concept experiment
To assess the biological performance of the porous biomaterial scaffold for bone repair, the scientists seeded bone-forming cells on the scaffold.
In their lab experiments, they found that the number of cells increased dramatically. After a week, the cells were evenly distributed on the scaffold – an indicator that the scaffold could promote appropriate cellular activities and eventually lead to tissue formation. Scientists also found that the presence of HA in the biomaterial significantly improved bone formation.
The biomaterial has also been tested for its tendency to elicit an inflammatory response, which is common after a biomaterial is implanted in the body. Using the real-time polymerase chain reaction, the scientists found that the level of expression of pro-inflammatory genes in human immune cells exposed to the biomaterial remained “relatively modest” compared to a control exposed to endotoxins, a compound known to stimulate the immune response, says Asst Prof Tay.
For example, expression of the IL6 gene in the biomaterials group was negligible and at least 50 times lower than that of immune cells exposed to endotoxins. This suggests that the risk of the biomaterial developed by NTU triggering an excessive acute inflammatory response is low.
Taken together, these results demonstrate the potential of biomaterial scaffolding, synthesized from discarded frog skin and fish scales, as a promising bone graft replacement material for bone repair and regeneration.
Ms. Chelsea Wan, Director of Jurong Frog Farm, said: âThe aquaculture industry is an important way to meet the growing global demand for safe and quality seafood, but one of the great challenges we face. are faced is the enormous waste and recycling of precious aquatic resources. In Singapore, the combined annual consumption of frog meat and fish is estimated to be around 100 million kilograms, making bullfrog skin and fish scales two of the largest secondary streams of aquaculture waste here. The integration of multiple seafood waste streams into one high-value product is a prime example of sustainable innovation for the aquaculture industry. “
In the future, the research team hopes to work with clinical and industrial partners on animal studies to find out how body tissue responds to this biomaterial in the long term, and the material’s ability to repair bone defects and wounds. cutaneous, as well as to bring the entire waste recovery technology pipeline closer to marketing.
This multidisciplinary research is conducted by MSE, SBS, NEWRI and the NTU Energy Research Institute.
Wang JK et al. Hybrid biocomposite derived from sustainable aquaculture for bone tissue engineering. Carpet. Sci. and Eng: C, Volume 126, July 2021, 112104. https://doi.org/10.1016 / j.msec.2021.112104
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