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Home>news>Big News>Recombinant collagen polypeptide as a versatile bone graft biomaterial

Recombinant collagen polypeptide as a versatile bone graft biomaterial

Dec 01 2020 share:

The current gold standard for bone grafting surgery includes autografts and allografts, although a growing demand exists to develop synthetic biomaterials for enhanced bio-integration in bone tissue engineering. In a new report now published on Nature Communications Materials, Hideo Fushimi and a research team in bioscience and engineering, and reconstructive biotechnology in Japan and the U.S., developed a biodegradable scaffold material using recombinant proteins or polypeptides as a source of hydrogel-based graft materials. The team used human type I collagen alpha 1 chain (abbreviated RCPhC1) as a source to develop the recombinant polypeptide and demonstrated the flexibility of the material to engineer ideal characteristics for bone grafts. The team also developed RCPhC1 bone grafts using a highly scalable, streamlined production protocol for the robust generation of mature bone tissue in the lab. The bone graft completely resorbed after tissue regeneration in a preclinical animal model for effective biological integration.                                                                                              

Bone tissue engineering with biomimetic, synthetic bone grafts

In this work, Fushimi et al. developed a synthetic bone graft material by using a recombinant protein abbreviated RCPhC1. The development provides a versatile source material to manufacture synthetic bone grafts via flexible engineering. Using preclinical studies in canine and rodent bone defect models, the team showed improved efficiency of the bone grafts to regenerate bone tissue with structural maturity.  In clinical orthopedics, the loss of tissue volume and function is a hallmark of injury, chronic inflammation, and metabolic and genetic disease. While bone tissue can actively regenerate via proliferation and osteogenic differentiation of mesenchymal stromal or stem cells, large bone defects require surgical interventions to repair and rebuild bones with bone graft materials.

Globally, orthopedic surgeons perform approximately 2.2 million bone graft procedures annually in an exceedingly costly global market. Human bone tissue is composed of organic extracellular matrix, crystallized calcium and phosphorous minerals that form hydroxyapatite. Bone graft materials can mimic the structure and biochemical composition of bone tissue. Orthopedic surgeons and researchers have used autologous bone grafts (cells and tissue obtained from the same individual) to repair bone defects due to mineral and immunological concerns, although complications at the sites of surgery can lead to alternative grafting methods such as allografts (cells and tissue obtained from a different individual). The newer development of biomimetic, synthetic biomaterials for bone tissue engineering addresses an urgent need in the healthcare industry to develop new graft materials without using human or animal tissue.

Engineering the recombinant polypeptide and the graft material

The team used type I collagen, abundantly expressed in connective tissues and interstitial membranes as a major organic component of bone tissue. The protein sequence of type I collagen plays an important role in establishing the mechanical strength of bone. The team first cloned the complimentary DNA (cDNA) – a DNA copy of a messenger RNA (mRNA) molecule that encodes the polypeptide (protein sequence) RCPhC1, into an expression vector. To accomplish this, they used a methylotrophic yeast species known as Pichia pastoris to transfer the sequence and generate master and working cell banks. The team confirmed the amino acid composition of the synthetic polypeptide and extensively characterized the product.

They then engineered the internal structure of the graft material to meet specific requirements of the target tissue. To generate a uniform pore structure, therefore, Fushimi et al. designed a thin-layer freeze casting (TLFC) protocol. The versatile freeze approach generated a large number of pores with thin walls to form an isotropic RCPhC1 scaffold with various internal structures. 

Dehydrothermal crosslinking profile and optimizing the material for bone regeneration

Fushimi et al. next subjected the material to dehydrothermal crosslinking treatment to test the effect of crosslinking temperature and duration on its composition. After testing the downstream product for microbial contamination, the temperature during the manufacturing process was confirmed to be effective for dry heat sterilization. Additional tests showed how the unique amino acid composition of the recombinant protein contributed to its robust hydrothermal crosslinking efficiency. The team next optimized the recombinant protein material for bone grafting by regulating the concentration of the polypeptide material and its freeze temperature based on the volume of graft-induced bone in a rat calvarial (skull) bone defect model. Four weeks after grafting the material in the animal model, the team used micro-computed tomography (micro CT)-based bone volume estimation.  The results indicated an optimal concentration of 7.5 percent RCPhC1, a freezing temperature of -40 to -60 degrees Celsius, and dehydrothermal crosslinking at 130?°C for 7?hours to be best suited for recombinant bone graft material manufacture.  


Robust regeneration of vital bone tissue in preclinical models with graft materials

Based on the experimentally verified optimal conditions, Fushimi et al. manufactured the RCPhC1 bone grafts with porous granules. Using the rat calvarial bone defect model, they showed how the bone graft robustly induced bone regeneration within the internal pore structure, while gradually degrading in vivo, to indicate biocompatibility and effective biointegration. They compared this outcome with a commercially available bovine (cow) decellularized cancellous bone xenograft and did not observe significantly greater bone regeneration. The team then tested the bone graft material in a canine preclinical model of tooth extraction to understand wound healing in the tooth socket, where the extraction socket treated with the bone graft showed improved bone formation at 12 weeks. By this time, as expected, the bone graft was largely replaced by bone tissue.

Outlook of synthetic scaffolds in bone tissue engineering

In this way, Hideo Fushimi and colleagues optimized a simple, but critical engineering process to regulate the solute concentration of a recombinant polypeptide of human type I collagen alpha I chain protein with targeted amino acid substitutions, abbreviated as RCPhC1. The team first implanted the construct in a rat calvarial bone defect model to understand the optimal engineering factors to manufacture the bone graft. They designed the bone graft material to support the migration of mesenchymal stem cells (MSCs) toward the defect area and provided a stimulating microenvironment for osteogenic differentiation. The bone graft material alone showed an ideal healing pattern in the absence of growth factors and stem cells to regenerate bone. The material can be used to generate tissue-specific medical devices and graft scaffolds with significant manufacturing versatility in bone tissue engineering.                                                                                                                          

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