Abstract

Throughout China, bone injuries, scoliosis, and bone defects have proven to be significant health challenges, heavily impacting the lives of citizens of the nation. These health conditions are often caused by trauma, infections, tumors, or congenital diseases, and result in serious consequences including functional impairment, chronic pain, inflammation, and restricted movement, creating severe impacts on a patient’s quality of life. Additionally, the treatment of scoliosis and the treatment of bone defects are essentially the same, both are to achieve bone healing, but the treatment of scoliosis spinal fusion is to heal two originally separated spinal bones into one, the treatment of bone defects is to repair or join the bone due to congenital or external force, and the two have the same need for rapid and safe bone formation. Although current treatment methods, such as autografts, allografts, and artificial bone materials are helpful, each faces critical limitations. Autografts face risks such as limited donor availability and the need for additional surgery. Allografts suffer risks of immune rejection and have high risks of infection. Further, artificial bone materials often have poor biocompatibility and lack bioactive components, such as cell factors[1, 2]. Today, artificial bone materials are the main source of bone repair materials and account for more than 90% of all bone repair materials. However, the inability to achieve a controlled release of cell factors through artificial bone materials poses serious dangers to users, such as risks of oncogenesis and ectopic ossification[3].

Our new bone repair study aims to develop and characterize a dual-cell factor release system that utilizes collagen-binding properties to overcome these dangerous limitations.

The two proteins, Bone Morphogenetic Protein 4 (BMP-4) [4] and Vascular Endothelial Growth Factor (VEGF) were selected as our target cell factors[5]. Out of ten collagen domains[6, 7], two domains were screened for minimal impact on both bioactivity and differential binding affinities. The collagen domain with high binding affinity was fused with BMP-4, while the domain with low binding affinity was fused with VEGF, enabling a sequential release in which VEGF is released first, followed by BMP-4. The dual cell factor release system provides a much more precise and effective control over critical cell factor release during tissue regeneration and repair, to effectively mimic the natural temporal expression of cell factors. The innovative dual-cell factor release design allows for the enhancement of repair efficacy while reducing the side effects associated with non-sequential release, to offer a far more efficient and safer bone repair solution. The proposed system illustrates significant potential for broad clinical applications, by elevating and refining the treatment of bone defects and associated conditions.

1 Inspiration

1.1 Inspiration from Real-World Problems

Our team’s project originates from a true story involving one of our classmates, and his younger brother. His brother often spent long hours looking down at the phone and sitting with poor posture, eventually leading to him developing scoliosis, a painful and debilitating condition. He found himself struggling with constant back pain, especially when he had to stand or sit for extended periods.

When his brother went to the hospital, the doctors informed him that spinal fusion surgery was the only option to correct his spine.

Spinal fusion surgery is a procedure used to join two or more vertebrae in the spine, eliminating motion between them. It is often performed to relieve pain caused by conditions such as degenerative disc disease, scoliosis, or spinal fractures. During the surgery, a portion of the existing spine, such as a damaged disc or bone spurs, may be removed to alleviate pressure on the nerves and create space. Bone grafts or bone repair materials are then used to fill this space. These grafts and materials act as a scaffold, promoting new bone growth and integration with the existing spine, which stabilizes the spine and ensures successful fusion.

His brother was very reluctant and distressed at the thought of undergoing such painful procedures. The psychological burden of facing such a daunting treatment left him feeling anxious and helpless.

As we saw the anguish and pain that his brother was going through, we realized that scoliosis is a fairly common condition among children in China. We consulted with doctors who confirmed that, unfortunately, the current treatments in China are quite limited and often involve invasive procedures like the one his brother was facing. The doctors mentioned a product called Infuse, which can promote rapid bone healing and reduce patient suffering. However, this product is not available in China and has significant side effects such as cancer and ectopic bone formation due to the rapid and large release of cell factors in a short period instead of gradual release.

Inspired by his brother’s situation and the need for a better solution, we decided to develop a bone repair material that can achieve the gradual release of cell factors. Our product is composed mainly of collagen and is designed to release VEGF and BMP4 cell factors in stages, promoting bone healing while minimizing side effects. Our product not only can rapidly promote bone healing in spinal fusion surgeries but also can be used in general fracture and bone defect scenarios. Through our innovation, we aim to help his brother and millions of other children in China suffering from bone issues, providing them with a safer and more effective treatment option and alleviating their pain and psychological distress.

Identifying the Problem

We noted significant issues with the current existing cell factor-based products, including poor usability and substantial problems during clinical application. This observation motivated us to delve deeper into understanding the underlying causes of these issues. Combining theoretical knowledge from coursework with a thorough review of relevant literature, we investigated the background of cell factor-based drug usage. This research revealed that the limited clinical use of these drugs is a widespread problem, particularly within local contexts.

Literature Review and Knowledge Application

Combining theoretical knowledge from coursework with a thorough review of relevant literature, we investigated the background of cell factor-based drug usage. This research revealed that the limited clinical use of these drugs is a widespread problem, particularly within local contexts.

Fig.1 The usage rate of infuse has been decreasing year by year since 2010.

Consultation with Experts

To gain a deeper understanding, we consulted with experts in the field. These discussions provided valuable insights into the challenges faced in clinical applications of cell factor-based therapies and confirmed the observations made during the hospital internship.

Team Brainstorming and Idealization

Armed with these insights and leveraging their academic knowledge, we engaged in brainstorming sessions. The team proposed innovative ideas to address the identified problems, leading to the development of a dual cell factor release system with collagen-binding properties. This system aims to enhance the clinical efficacy and safety of cell factor-based treatments, offering a promising solution to the observed challenges.

1.2 Inspiration from Previous iGEM Projects

iGEM is built on a tradition of innovation, where teams often draw inspiration from the successes and challenges faced by past participants. After reviewing several iGEM projects, we recognized the critical role that collagen plays in bone repair. Projects like NYU-Abu-Dhabi 2023 and CCA San Diego 2021 demonstrated the potential of collagen in developing biomaterials for tissue engineering. These teams emphasized the importance of collagen's structural stability and its application in regenerative medicine. Inspired by their findings, we chose collagen as our material matrix and decided to use collagen-binding domains as tags for our cell factors to enhance bone repair.

2 Issue

2.1 The Severity of Bone Issues in Chinese Children

Chinese children commonly face skeletal issues, with approximately 5 million children suffering from varying degrees of congenital scoliosis. Many acquired bad habits and accidents, such as prolonged use of smartphones with a lowered head[8], improper sitting posture[9], and accidental falls[10], can also lead to skeletal problems and exacerbate the severity of congenital scoliosis.

Data from domestic studies and surveys underscore the severity of these issues. For instance, a survey of schoolchildren revealed high incidences of spinal deformities and bone injuries, directly linked to their sedentary lifestyle and academic pressures.

2.2 Current Treatment’s Problems

Traditional bone repair materials, such as autografts, allografts, and metallic materials, face significant limitations. Autografts, though highly biocompatible and effective in osteogenesis, are limited by donor availability and the invasive nature of harvesting, leading to donor site morbidity. Allografts, while more available, pose risks of immune rejection and infection, with moderate biocompatibility and osteogenesis. Xenografts face high immune rejection rates and poor biocompatibility, limiting their use. Metallic materials like medical stainless steel and titanium alloys offer high mechanical strength but suffer from poor biocompatibility and osteogenesis, making them less ideal for seamless integration with natural bone tissue. Additionally, these traditional materials lack active biological components[11].

Active bone repair materials, such as bioactive ceramics and synthetic polymers, have been developed to address some of these limitations by incorporating bioactive components. However, these materials also face significant challenges. For instance, while materials like β-TCP[12] and PEEK[13, 14] demonstrate good biocompatibility and osteogenesis, they often lack sufficient mechanical strength for load-bearing applications. Furthermore, the inclusion of cell factors in some synthetic polymers, such as chitosan hydrogels, has not resolved the fundamental issue of controlled release[15]. The inability to achieve sustained, controlled release of cell factors results in an initial burst release that can lead to severe side effects, including oncogenesis and ectopic bone formation.[16, 17] These drawbacks underscore the need for innovative solutions that can provide both mechanical strength and controlled bioactivity to enhance bone repair outcomes.

Fig.2 Current bone repair material and current materia drawbacks

What if ...?

What if TestDaily-I-Beijing could develop a collagen-based bone repair material that systematically releases VEGF and BMP4 cytokines in stages, thereby promoting bone healing and reducing the painful side effects associated with traditional scoliosis treatments?

3 Project

3.1 Project Overview

Bone repair is a complex, systematic process, similar to developing a suburban area. Just like roads are essential for transporting materials before constructing buildings, bone repair starts with promoting blood vessel formation at the defect site, ensuring the smooth transport of oxygen and nutrients. Next, osteoblasts produce hydroxyapatite for bone formation.

But how do we achieve a gradient release of these two cell factors?

In our cell biology course, we learned that collagen-binding domains are widespread in nature and allow proteins like collagenases to bind effectively to collagen. Collagen is an excellent scaffold material for bone repair, which led us to an idea: could we fuse collagen-binding domains with cell factors to achieve controlled, sustained release?

We discussed this idea with experts in the field. After thorough consultations, they confirmed the feasibility of our approach. Their positive feedback reinforced our confidence and encouraged us to further explore and develop this innovative technology. By combining collagen-binding domains with cell factors, we aim to create a more effective and safe bone repair solution for many patients in need.

Our study aims to develop and characterize a dual cell factor release system with collagen-binding properties, selecting BMP-4 and VEGF as the target cell factors.

The design concept is based on a sequential release strategy to mimic the natural bone healing process.

The collagen domain with high binding affinity was fused with BMP-4, while the domain with low binding affinity was fused with VEGF. This strategic fusion allowed us to achieve the desired sequential release: VEGF, which is essential for angiogenesis, is released first to ensure the formation of a vascular network, followed by the release of BMP-4, which promotes the differentiation of osteoblasts and bone formation.

Fig.3 VEGF(left) for angiogenesis and BMP-4(right) for bone formation

This innovative approach provides precise and effective control over cell factor release, enhancing the overall efficacy and safety of bone repair. By mimicking the natural temporal expression of cell factors during tissue regeneration, this system offers significant potential for improving clinical outcomes in bone repair and regeneration.

3.2 Selection of Collagen Binding Domain

Based on our discussions with an expert, it was determined that selecting appropriate collagen binding domains is critical to the functionality of our product. From 8 collagen domains, we screened out 2 domains that have minimal impact on bioactivity and exhibit different binding affinities. This screening was crucial to ensure that the selected domains could effectively control the release timing of the cell factors without compromising their biological functions.

Fig.4 Effects of different collagen binding domains on EGFP properties

Fig.5 Binding ability of different collagen binding modules to collagen

3.3 Construction of Fusion Proteins

The expert emphasized the importance of strategic protein fusion to achieve sequential release of cell factors. The collagen domain with high binding affinity was fused with BMP-4, while the domain with low binding affinity was fused with VEGF. This strategic fusion allowed us to achieve the desired sequential release: VEGF, which is essential for angiogenesis, is released first to ensure the formation of a vascular network, followed by the release of BMP-4, which promotes the differentiation of osteoblasts and bone formation.

Based on the selected collagen-binding tags, we constructed two fusion protein plasmids as shown in the figure. The first plasmid, pET-28a-CBD-VEGF, incorporates the CBD MMPs tag fused with VEGF. The CBD MMPs tag was chosen for its collagen-binding properties, and the fusion aims to facilitate the controlled release of VEGF from collagen-based materials. The second plasmid, pET-28a-FTD-BMP4, features the FTD tag fused with BMP4. The FTD tag, identified for its strong binding affinity to collagen, ensures effective binding and gradual release of BMP4 in the collagen matrix.

Fig.6 cell factor plasmids constructed in this study

3.4 Preparation of Collagen Hydrogel

In our expert consultation, it was highlighted that the preparation and characterization of the hydrogel scaffold are paramount for ensuring its suitability for bone repair. We successfully prepared a suitable hydrogel scaffold and characterized its mechanical properties, rheology, and biocompatibility. After testing various collagen concentrations, we determined that the 20% collagen scaffold offered the optimal combination of tensile strength, viscosity, and biocompatibility, making it ideal for bone repair and regeneration applications.

3.5 Characterization of Biological Activity and Binding Activity

According to the expert’s guidance, it was essential to verify the biological activity and binding efficacy of our constructs. The collagen domain with high binding affinity was fused with BMP-4, while the domain with low binding affinity was fused with VEGF. This strategic fusion allowed us to achieve the desired sequential release: VEGF, which is essential for angiogenesis, is released first to ensure the formation of a vascular network, followed by the release of BMP-4, which promotes the differentiation of osteoblasts and bone formation.

References