1 DBTL Cycle 1: Screening Collagen-Binding Domains (CBDs)

We constructed several CBD-EGFP fusion proteins and expressed them in bacterial systems to analyze their activity.

We first commissioned a company to synthesize the gene sequences of each CBD, and then used seamless cloning to connect the gene sequences of each CBD with the EGFP sequence already in the laboratory.

In this stage, the colony PCR results show successful amplification of the eight different CBD-EGFP fusion proteins, confirming the correct construction of the engineered strains. The bands visible in the gel correspond to the expected sizes of the fusion proteins:

The PCR bands in the gel align well with these predicted sizes, demonstrating that each fusion construct was correctly assembled, which is crucial for proceeding with protein expression and purification.

Fig1.1 Plasmid design

We then used these E. coli to express proteins and measured the fluorescence value of each protein host bacteria at the same bacterial concentration. Some of the fusion proteins were then purified by Ni-NTA for further analysis.

The fluorescence activity of each fusion protein was tested to evaluate whether the CBDs affected EGFP activity. The relative fluorescence values of the eight CBD-EGFP fusion proteins were measured to assess the impact of the collagen-binding domains (CBDs) on EGFP activity. As shown in the graph:

Overall, the majority of CBDs retained sufficient EGFP activity, which confirms that they can be used in further experiments for controlled release without significantly impairing the functionality of the fusion proteins. These results support the selection of CBDs that balance collagen binding with maintaining protein bioactivity.

Fig1.2 The fluorescence activity of each fusion protein

Binding affinity tests (via microscale thermophoresis) were also conducted to determine which CBDs had high binding affinity (for BMP-4) and low binding affinity (for VEGF). Based on the binding experiment shown in the graph, the results illustrate the relative binding affinities of different CBD-EGFP fusion proteins to collagen, with the binding affinity represented by the dissociation constant (Kd). The midpoint of each curve on the x-axis corresponds to the Kd value, indicating how tightly the CBDs bind to collagen.

These Kd values highlight that FTD is the most suitable for applications requiring strong binding, while CBD MMPs may be used in situations where weaker, more transient binding is needed.

2 DBTL Cycle 2: Developing the Dual Cell Factor Release System

FTD-BMP-4 proved more challenging. The presence of multiple disulfide bonds in BMP-4 led to the formation of inclusion bodies when expressed in BL21, which made the protein insoluble and inactive. To overcome this, we explored various optimized bacterial strains designed to facilitate proper folding of disulfide bond-containing proteins.

Several strains were tested:

The culmination of this stage was obtaining both fusion proteins in their active, soluble forms, laying the foundation for further testing of their controlled release and biological activity in subsequent phases. In Fig3.2, lanes 1 to 4, 5 to 8, and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after disruption, respectively. Each group consists of four lanes, and the order within each group is BL21, ArcticExpress (DE3) pRARE, Shuffle T7-B, Origami (DE3)

Fig2.1 Results of expression system optimization a) SDS-PAGE; b) semi-quantitative analysis (Lanes 1 to 4, 5 to 8, and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after disruption, respectively. Each group consists of four lanes, and the order within each group is BL21, ArcticExpress (DE3) pRARE, Shuffle T7-B, Origami (DE3))

In the gel (left) and the bar chart (right), ArcticExpress shows significantly improved soluble protein expression compared to other strains like BL21, which primarily produces inclusion bodies. This makes ArcticExpress the optimal choice for expressing soluble and active FTD-BMP-4, ensuring that a higher percentage of the total protein is in the desired soluble form.

In this experiment, the release profiles of CBD MMPs-VEGF and FTD-BMP-4 from collagen hydrogels were monitored over time. The two fusion proteins were incorporated into collagen hydrogels, and their release was quantified using ELISA assays. The hydrogels were incubated in PBS at 37°C, with supernatant samples collected at regular intervals to measure the amount of protein released. After each sampling, fresh PBS was added to the hydrogels to maintain consistent conditions throughout the experiment.

Fig2.2 Time-Dependent Release Profiles of CBD MMPs-VEGF and FTD-BMP-4 from Collagen Hydrogel

The results reveal a significant difference in the release kinetics between the two cell factors. CBD MMPs-VEGF showed a rapid and early release pattern, starting with an accelerated increase in the release rate during the first 10 days. By day 10, more than 50% of the VEGF had already been released from the hydrogel. This sharp increase continued, reaching approximately 75% release by day 20. Following this, the release rate gradually slowed, with the curve plateauing as nearly 100% of the VEGF was released by day 30. The release curve of VEGF shows an initial rapid release phase, followed by a stabilization period where the release rate gradually levels off.This fast release behavior can be attributed to the lower binding affinity of the CBD MMPs domain, allowing VEGF to dissociate from the collagen matrix more quickly.

In contrast, FTD-BMP-4 exhibited a much slower and more controlled release. During the first 10 days, less than 20% of BMP-4 was released, indicating a more gradual release process. The release steadily increased over time, with the curve following a linear trend. By day 20, around 30% of BMP-4 had been released, and by day 30, approximately 50% was released. The release continued at a steady pace, reaching close to 100% around day 40. This extended release profile reflects the higher binding affinity of the FTD domain to collagen, which ensures a slower and sustained release of BMP-4 over the course of the experiment.

These contrasting release profiles demonstrate the effectiveness of using different collagen-binding domains to control the timing and rate of cell factor release, with CBD MMPs facilitating a faster release for VEGF and FTD ensuring a prolonged release for BMP-4.These results confirm that the controlled, sequential release of the two cell factors can be achieved by leveraging the different binding affinities of the CBDs, with CBD MMPs-VEGF releasing faster to initiate angiogenesis, followed by the slower release of FTD-BMP-4 to promote osteogenesis.

The tube formation assay is a widely used in vitro method for assessing the ability of endothelial cells, such as HUVECs (Human Umbilical Vein Endothelial Cells), to form capillary-like structures, mimicking the process of angiogenesis. This assay is particularly useful for evaluating the angiogenic potential of cell factors, small molecules, or other treatments.

In the tube formation assay, human umbilical vein endothelial cells (HUVECs) were seeded onto a Matrigel-coated plate to facilitate tube formation. The experimental group was treated with CBD MMPs-VEGF at a final concentration of 100 ng/ml, while the control group received no VEGF. The cells were incubated at 37°C with 5% CO₂ for 4-6hours, during which tube formation was observed. The formation of capillary-like structures was monitored under a microscope to assess angiogenesis. The extent of tube formation between the experimental and control groups was compared to determine the impact of CBD MMPs-VEGF on promoting angiogenesis.

Fig2.3 Comparison of Tube Formation in HUVECs a) Control; b)CBD MMPs-VEGF Treatment

Image a (Without CBD MMPs-VEGF): In the control group, where no CBD MMPs-VEGF was added, the HUVECs exhibited weak angiogenic activity. Only a few scattered cell connections were observed, with minimal evidence of network formation. Quantitatively, the tube formation rate (measured by parameters such as total tube length, number of branch points, and closed loops or tube closure rate) was significantly reduced. The tube closure rate, which measures the percentage of formed capillary-like loops, was close to zero, reflecting the lack of organized tube structures. This result confirms that without the presence of VEGF, the cells are unable to initiate or sustain effective angiogenesis.

Image b (CBD MMPs-VEGF at 100 ng/ml): In the experimental group treated with 100 ng/ml CBD MMPs-VEGF, the HUVECs showed robust tube formation, with clearly defined and well-organized capillary-like structures. Quantitative analysis revealed a dramatic increase in several angiogenesis indicators. The tube closure rate increased to approximately 70-80%, indicating a high number of complete tube loops. Additionally, the total tube length and number of branch points were significantly higher compared to the control, demonstrating enhanced network complexity and cell connectivity. These findings suggest that the addition of VEGF significantly promoted angiogenesis, with the cells forming a dense and interconnected tubular network, characteristic of active capillary formation.

This comparison highlights the essential role of CBD MMPs-VEGF in promoting angiogenesis, as quantified by standard tube formation metrics. The increased tube closure rate, total tube length, and branch points in the experimental group strongly indicate that the VEGF produced in the system retains its biological function and effectively stimulates endothelial cell organization and network formation.The comparison between these two images confirms that the CBD MMPs-VEGF produced in this study retains biological activity and effectively stimulates tube formation, validating its functional ability to promote angiogenesis.

In our osteogenic differentiation experiments, MC3T3-E1 cells, a well-established pre-osteoblast cell line derived from mouse calvaria, were used. These cells are commonly employed in bone research because they have the inherent capacity to differentiate into mature osteoblasts when cultured under osteogenic conditions. During the early stages of osteogenic differentiation, MC3T3-E1 cells express alkaline phosphatase (ALP), which is critical for mineralization. ALP staining can be used to measure the early osteogenic activity in these cells. As differentiation progresses, MC3T3-E1 cells begin to deposit calcium in the extracellular matrix, which is a key indicator of bone formation. This can be detected using Alizarin Red staining, which binds specifically to the calcium deposits and allows for the visualization of mineralization. Thus, by using both ALP staining and Alizarin Red staining, the osteogenic differentiation process of MC3T3-E1 cells can be effectively tracked from early osteoblast activity (ALP expression) to late-stage bone formation (calcium deposition).

Together, ALP staining and Alizarin Red staining offer a comprehensive view of osteogenic differentiation. ALP staining identifies early differentiation events, while Alizarin Red staining confirms matrix mineralization, showing that the cells are functionally mature osteoblasts capable of bone formation.

For the ALP staining, cells were cultured for 7 and 14 days, then washed with PBS and fixed with 4% paraformaldehyde for 10-15 minutes at room temperature. After washing with PBS, the cells were incubated with ALP staining solution (e.g., BCIP/NBT) for 30-60 minutes at 37°C. ALP activity, indicating early osteogenic differentiation, was visualized as blue-purple staining under a microscope. For the Alizarin Red staining, cells were cultured for 21 and 28 days, washed with PBS, and fixed with 4% paraformaldehyde for 10-15 minutes. After washing with distilled water, Alizarin Red S solution (pH 4.1-4.3) was applied for 20-30 minutes to detect calcium deposits. The cells were then rinsed with distilled water, and red staining was observed to indicate matrix mineralization, a marker of mature osteoblast activity.

Fig2.4 ALP and Alizarin Red Staining of MC3T3-E1 Cells at Different Time Points During Osteogenic Differentiation

ALP Staining Results (Day 7 and Day 14): From the images provided, on Day 7 (top-left images), the ALP staining shows minimal blue-purple staining, suggesting that osteogenic differentiation had only just begun. By Day 14 (bottom-left images), there is a noticeable increase in blue-purple staining, indicating elevated ALP activity, which correlates with increased osteoblast differentiation. The stronger ALP activity seen on Day 14 demonstrates that the cells are undergoing early osteogenic differentiation, which is being effectively stimulated by the BMP-4 cell factor, confirming its biological activity in promoting bone formation.

Alizarin Red Staining Results (Day 21 and Day 28): In the Day 21 images (top-right), there are initial signs of mineralization with scattered red deposits, indicating the onset of calcium deposition. By Day 28 (bottom-right), the staining intensity has significantly increased, with larger and more densely stained red areas, showing substantial mineralization of the extracellular matrix. This confirms the later-stage osteogenic activity, as BMP-4 continues to drive the maturation of osteoblasts and the deposition of calcium, which is a hallmark of bone tissue formation.

These results confirm that the BMP-4 produced in our system retains its biological activity, as it successfully stimulates both early (ALP activity) and late (mineralization) osteogenic differentiation.

3 DBTL Cycle 3: Development and Validation of the Collagen Hydrogel System

A collagen hydrogel system was designed to sequentially release two cell factors—VEGF and BMP-4—for bone tissue regeneration. Collagen was chosen as the base material due to its biocompatibility, biodegradability, and bioactivity, promoting cell adhesion and proliferation. The engineered Type I collagen was expressed using E. coli, and the hydrogel allowed for the rapid release of VEGF to stimulate angiogenesis, followed by the slower release of BMP-4 to support osteogenesis.