Periodic Reporting for period 1 - AffordBoneS (Development of personalized and affordable multi-substituted calcium phosphate based scaffolds for bone augmentation applications)
Reporting period: 2022-10-01 to 2024-09-30
1. Summary of the context and overall objectives of the project
According to the World Health Organization (WHO), less than half of the world’s population has access to essential healthcare services, highlighting the need for affordable solutions (1). The AffordBoneS project aimed to address this by developing personalized and affordable scaffolds for bone augmentation procedures. This would allow more people to access these treatments and improve their quality of life. The project focused on creating cost-effective materials for bone augmentation that could be used by a larger population. The solution involved fabricating customizable, biomimetic scaffolds tailored to each patient’s jaw bone defect. The three main research objectives were to produce highly porous, multi-substituted calcium phosphate (mCaP) scaffolds suitable for bone regeneration.
Objective 1: Designing, fabrication and characterization of mCaP Scaffolds (2).
Objective 2: Biological characterization of mCaP Scaffolds by using human stem cells.
Objective 3: Demonstrating personalized mCaP Scaffolds.
2. Introduction
In Europe alone, it is estimated that 1.5 million bone augmentation procedures are needed each year (3). These procedures are crucial for dental implants when patients don’t have enough bone to secure the implant. The AffordBoneS project aimed to create affordable and customizable solutions for these procedures using 3D-printed calcium phosphate scaffolds enhanced with beneficial ions.This innovative approach offers several advantages over current methods, such as eliminating the need for artificial growth factors, reducing costs through mass customization, and avoiding the need for a second surgery to remove the scaffold. The project brought together experts from various fields, including material engineers, biologists, and companies, to improve bone regeneration techniques.
Large bone defects (greater than 2 cm) cannot heal on their own and require scaffolds to guide and support the regeneration process. However, challenges remain in bone tissue engineering, such as reducing the use of growth factors, minimizing immune responses and scaffold rejection, improving blood vessel formation (vascularization), and enhancing mechanical strength (4).While growth factors are commonly used with calcium phosphate ceramics to promote bone growth, concerns have been raised about their safety, including risks of unwanted bone formation, dosage issues, instability, high costs, and potential long-term side effects (5, 6). A promising alternative is to incorporate key elements found in natural bone, such as strontium (Sr2+), magnesium (Mg2+), sodium (Na+), and zinc (Zn2+), into the scaffolds (7).Preliminary studies have shown that scaffolds containing these ions significantly improve bone regeneration compared to those without (8). Despite these advancements, scalable manufacturing and commercialization of these scaffolds remain challenge. Additive manufacturing (3D printing) offers a solution to these challanges by creating scaffolds with the precise microstructure and mechanical strength needed for effective bone regeneration.
References:
1. www.who.int/news/item/13-12-2017-world-bank-and-who-half-the-world-lacks-access-to-essential-health-services-100-million-still-pushed-into-extreme-poverty-because-of-health-expenses
2. A. Ressler et al., Vat photopolymerization of biomimetic bone scaffolds based on Mg, Sr, Zn-substituted hydroxyapatite: Effect of sintering temperature, Ceramics International 50(2024) 27403-27415. https://doi.org/10.1016/j.ceramint.2024.05.038
3. A. Hoornaert and P. Layrolle, 2020: Bone regenerative issues related to bone grafting biomaterials, in Dental Implants and Bone Grafts, Chapter 8, 207-215. https://doi.org/10.1016/B978-0-08-102478-2.00009-X
4. S. Kashte et al., Artificial Bone via Bone Tissue Engineering: Current Scenario and Challenges, Tissue Engineering and Regenerative Medicine 14 (2017) 14 1-4. https://doi.org/10.1007/s13770-016-0001-6
5. S. Bose et al., Understanding of dopant-induced osteogenesis and angiogenesis in calcium phosphate ceramics, Trends in Biotechnology 10 (2013) 594-605. https://doi.org/10.1016/j.tibtech.2013.06.005
6. N. Goonoo and A. Bhaw-Luximon, Mimicking growth factors: role of small molecule scaffold additives in promoting tissue regeneration and repair, RSC Advances, 9 (2019) 18124. https://doi.org/10.1039/C9RA02765C
7. T. Cordonnier et al., Biomimetic Materials for Bone Tissue Engineering – State of the Art and Future Trends, Advanced Engineering Materials, 13 (2011) 5. https://doi.org/10.1002/adem.201080098
8. A. Ressler et al., Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold, Carbohydrate Polymers 277 (2022) 118883. https://doi.org/10.1016/j.carbpol.2021.118883
According to the World Health Organization (WHO), less than half of the world’s population has access to essential healthcare services, highlighting the need for affordable solutions (1). The AffordBoneS project aimed to address this by developing personalized and affordable scaffolds for bone augmentation procedures. This would allow more people to access these treatments and improve their quality of life. The project focused on creating cost-effective materials for bone augmentation that could be used by a larger population. The solution involved fabricating customizable, biomimetic scaffolds tailored to each patient’s jaw bone defect. The three main research objectives were to produce highly porous, multi-substituted calcium phosphate (mCaP) scaffolds suitable for bone regeneration.
Objective 1: Designing, fabrication and characterization of mCaP Scaffolds (2).
Objective 2: Biological characterization of mCaP Scaffolds by using human stem cells.
Objective 3: Demonstrating personalized mCaP Scaffolds.
2. Introduction
In Europe alone, it is estimated that 1.5 million bone augmentation procedures are needed each year (3). These procedures are crucial for dental implants when patients don’t have enough bone to secure the implant. The AffordBoneS project aimed to create affordable and customizable solutions for these procedures using 3D-printed calcium phosphate scaffolds enhanced with beneficial ions.This innovative approach offers several advantages over current methods, such as eliminating the need for artificial growth factors, reducing costs through mass customization, and avoiding the need for a second surgery to remove the scaffold. The project brought together experts from various fields, including material engineers, biologists, and companies, to improve bone regeneration techniques.
Large bone defects (greater than 2 cm) cannot heal on their own and require scaffolds to guide and support the regeneration process. However, challenges remain in bone tissue engineering, such as reducing the use of growth factors, minimizing immune responses and scaffold rejection, improving blood vessel formation (vascularization), and enhancing mechanical strength (4).While growth factors are commonly used with calcium phosphate ceramics to promote bone growth, concerns have been raised about their safety, including risks of unwanted bone formation, dosage issues, instability, high costs, and potential long-term side effects (5, 6). A promising alternative is to incorporate key elements found in natural bone, such as strontium (Sr2+), magnesium (Mg2+), sodium (Na+), and zinc (Zn2+), into the scaffolds (7).Preliminary studies have shown that scaffolds containing these ions significantly improve bone regeneration compared to those without (8). Despite these advancements, scalable manufacturing and commercialization of these scaffolds remain challenge. Additive manufacturing (3D printing) offers a solution to these challanges by creating scaffolds with the precise microstructure and mechanical strength needed for effective bone regeneration.
References:
1. www.who.int/news/item/13-12-2017-world-bank-and-who-half-the-world-lacks-access-to-essential-health-services-100-million-still-pushed-into-extreme-poverty-because-of-health-expenses
2. A. Ressler et al., Vat photopolymerization of biomimetic bone scaffolds based on Mg, Sr, Zn-substituted hydroxyapatite: Effect of sintering temperature, Ceramics International 50(2024) 27403-27415. https://doi.org/10.1016/j.ceramint.2024.05.038
3. A. Hoornaert and P. Layrolle, 2020: Bone regenerative issues related to bone grafting biomaterials, in Dental Implants and Bone Grafts, Chapter 8, 207-215. https://doi.org/10.1016/B978-0-08-102478-2.00009-X
4. S. Kashte et al., Artificial Bone via Bone Tissue Engineering: Current Scenario and Challenges, Tissue Engineering and Regenerative Medicine 14 (2017) 14 1-4. https://doi.org/10.1007/s13770-016-0001-6
5. S. Bose et al., Understanding of dopant-induced osteogenesis and angiogenesis in calcium phosphate ceramics, Trends in Biotechnology 10 (2013) 594-605. https://doi.org/10.1016/j.tibtech.2013.06.005
6. N. Goonoo and A. Bhaw-Luximon, Mimicking growth factors: role of small molecule scaffold additives in promoting tissue regeneration and repair, RSC Advances, 9 (2019) 18124. https://doi.org/10.1039/C9RA02765C
7. T. Cordonnier et al., Biomimetic Materials for Bone Tissue Engineering – State of the Art and Future Trends, Advanced Engineering Materials, 13 (2011) 5. https://doi.org/10.1002/adem.201080098
8. A. Ressler et al., Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold, Carbohydrate Polymers 277 (2022) 118883. https://doi.org/10.1016/j.carbpol.2021.118883
Objective 1:
In response to the urgent demand for innovative bone regeneration solutions, the focus of this objective was placed on the development and characterization of calcium phosphate scaffolds substituted with magnesium (Mg), strontium (Sr), and zinc (Zn) to replicate the trabecular architecture of cancellous bone. Porous scaffolds mimicking the natural bone structure were additively manufactured from photosensitive ceramic suspensions using vat photopolymerization and digital light processing. The impact of the selected trace elements and the sintering temperature was investigated in relation to the obtained crystalline phase content, microstructure, elemental distribution, thermal stability, and mechanical properties. After sintering, hydroxyapatite and β-tricalcium phosphate were detected as a result of the added trace elements in the calcium-deficient hydroxyapatite used as a starting powder. The microstructure characteristics of obtained scaffolds closely resemble the morphology of cancellous bone tissue, while the mechanical properties of the scaffolds were found to be in line with those typically observed in trabecular bone (2).
Objective 2:
Four different types of scaffolds with varying pore sizes were created to find out which one would be most effective for bone regeneration. The average pore sizes were: 267.64 µm for Scaffold 1, 396.54 µm for Scaffold 2, 707.33 µm for Scaffold 3, and 850.80 µm for Scaffold 4. The biological tests included analyzing cell number, alkaline phosphatase (ALP) expression, live/dead cell assays, and staining for collagen I and osteocalcin bone markers, both in standard and bone-growing (osteogenic) conditions. Scaffold 2 had the best chaarcteristics, making it the best candidate for further experiments.
Next, Scaffold 2 was tested with non-substituted calcium phosphate and calcium phosphate with low and high amounts of zinc, strontium, and magnesium ions. Initial results showed that adding these ions reduced cell attachment to the scaffolds. To understand why, further tests will measure the zeta potential of the scaffolds. One possible reason for this could be changes in the material during heating, which might affect how cells attach.
Objective 3:
During Objective 1, challenges with cleaning the complex printed structures were encountered. These challenges increased as the scaffolds became larger and more intricate. Before addressing complex jawbone defects based on real case models (Objective 3), the most effective cleaning method needed to be evaluated. Although the cleaning study was not part of the original AffordBoneS project plan, the difficulties in cleaning highly complex scaffolds and the lack of existing research on this topic made it clear that an in-depth study was necessary (9).
Given the limitations of conventional spray cleaning, alternative cleaning methods were investigated in this study. The effectiveness of dibasic ester (DBE) and LithaSol 80, combined with ultrasonic and soaking techniques, was tested to thoroughly remove the viscous residual slurry. The scaffolds were soaked in cleaning solutions at different temperatures for 24 hours. Based on the results, 50 °C was chosen for further analysis using both soaking and ultrasonic cleaning methods. The cross-sectional images showed that at least 48 hours of soaking or 30 minutes of ultrasonic cleaning were required for effective cleaning. Smoother surfaces were observed in scaffolds cleaned with LithaSol 80, while those treated with DBE showed contracted pores and a peeling effect, indicating that DBE was more aggressive. A significant difference in mass loss was observed between scaffolds treated with LithaSol 80 and DBE, with DBE causing higher mass loss, suggesting it affected both uncured and cured slurry. The results indicated that while DBE was more effective in cleaning, LithaSol 80 was better for maintaining structural integrity when combined with soaking.
During the AffordBoneS project, cleaning challenges prevented the successful fabrication of personalized scaffolds based on real case models. Moving forward, the goal is to revisit this objective to ensure that future scaffold production meets the personalized specifications of real-life clinical cases while preserving the necessary structural and material properties for successful application.
References:
9.A. Ressler et al., Cleaning strategies for 3D-printed porous scaffolds used for bone regeneration fabricated via ceramic vat photopolymerization, Ceramics International (2024) In Press. https://doi.org/10.1016/j.ceramint.2024.10.160
In response to the urgent demand for innovative bone regeneration solutions, the focus of this objective was placed on the development and characterization of calcium phosphate scaffolds substituted with magnesium (Mg), strontium (Sr), and zinc (Zn) to replicate the trabecular architecture of cancellous bone. Porous scaffolds mimicking the natural bone structure were additively manufactured from photosensitive ceramic suspensions using vat photopolymerization and digital light processing. The impact of the selected trace elements and the sintering temperature was investigated in relation to the obtained crystalline phase content, microstructure, elemental distribution, thermal stability, and mechanical properties. After sintering, hydroxyapatite and β-tricalcium phosphate were detected as a result of the added trace elements in the calcium-deficient hydroxyapatite used as a starting powder. The microstructure characteristics of obtained scaffolds closely resemble the morphology of cancellous bone tissue, while the mechanical properties of the scaffolds were found to be in line with those typically observed in trabecular bone (2).
Objective 2:
Four different types of scaffolds with varying pore sizes were created to find out which one would be most effective for bone regeneration. The average pore sizes were: 267.64 µm for Scaffold 1, 396.54 µm for Scaffold 2, 707.33 µm for Scaffold 3, and 850.80 µm for Scaffold 4. The biological tests included analyzing cell number, alkaline phosphatase (ALP) expression, live/dead cell assays, and staining for collagen I and osteocalcin bone markers, both in standard and bone-growing (osteogenic) conditions. Scaffold 2 had the best chaarcteristics, making it the best candidate for further experiments.
Next, Scaffold 2 was tested with non-substituted calcium phosphate and calcium phosphate with low and high amounts of zinc, strontium, and magnesium ions. Initial results showed that adding these ions reduced cell attachment to the scaffolds. To understand why, further tests will measure the zeta potential of the scaffolds. One possible reason for this could be changes in the material during heating, which might affect how cells attach.
Objective 3:
During Objective 1, challenges with cleaning the complex printed structures were encountered. These challenges increased as the scaffolds became larger and more intricate. Before addressing complex jawbone defects based on real case models (Objective 3), the most effective cleaning method needed to be evaluated. Although the cleaning study was not part of the original AffordBoneS project plan, the difficulties in cleaning highly complex scaffolds and the lack of existing research on this topic made it clear that an in-depth study was necessary (9).
Given the limitations of conventional spray cleaning, alternative cleaning methods were investigated in this study. The effectiveness of dibasic ester (DBE) and LithaSol 80, combined with ultrasonic and soaking techniques, was tested to thoroughly remove the viscous residual slurry. The scaffolds were soaked in cleaning solutions at different temperatures for 24 hours. Based on the results, 50 °C was chosen for further analysis using both soaking and ultrasonic cleaning methods. The cross-sectional images showed that at least 48 hours of soaking or 30 minutes of ultrasonic cleaning were required for effective cleaning. Smoother surfaces were observed in scaffolds cleaned with LithaSol 80, while those treated with DBE showed contracted pores and a peeling effect, indicating that DBE was more aggressive. A significant difference in mass loss was observed between scaffolds treated with LithaSol 80 and DBE, with DBE causing higher mass loss, suggesting it affected both uncured and cured slurry. The results indicated that while DBE was more effective in cleaning, LithaSol 80 was better for maintaining structural integrity when combined with soaking.
During the AffordBoneS project, cleaning challenges prevented the successful fabrication of personalized scaffolds based on real case models. Moving forward, the goal is to revisit this objective to ensure that future scaffold production meets the personalized specifications of real-life clinical cases while preserving the necessary structural and material properties for successful application.
References:
9.A. Ressler et al., Cleaning strategies for 3D-printed porous scaffolds used for bone regeneration fabricated via ceramic vat photopolymerization, Ceramics International (2024) In Press. https://doi.org/10.1016/j.ceramint.2024.10.160
Up to 50% of dental implants require a bone augmentation procedure before they can be fixed in place (10). Currently, this often involves using a titanium mesh to support a bioactive ceramic paste or bone from the patient, which helps regenerate the bone defect. However, this method requires a second surgery to remove the metal mesh, which increases costs and poses significant risks to the patient’s well-being (11).The European market for dental bone grafts is expected to grow significantly, from €162.24 million in 2021 to €255.40 million in 2026 (10). Meeting this market demand effectively is crucial. Developing and commercializing new scaffolds can take 10-15 years due to the gap between academic research, industry, and clinical trials. Collaboration among different stakeholders can help shorten and streamline this process.
The AffordBoneS project focuses on creating affordable and customizable scaffolds for bone augmentation procedures to be used before dental implants placement. These scaffolds could also be used for other bone regeneration applications, expanding their potential market. Significant progress was made in the AffordBoneS project using ceramic vat photopolymerization to create complex bone scaffolds. These scaffolds closely mimic the structure of natural spongy bone, with pore sizes ranging from 10 to 900 micrometers and a total porosity of about 76%, making them ideal for bone regeneration. However, cleaning the uncured ceramic material from these intricate structures was challenging. To address this, various cleaning methods were tested. Traditional spray cleaning was ineffective, so alternative methods using soaking and ultrasonic cleaning with dibasic ester (DBE) and LithaSol 80 were explored. By optimizing the cleaning temperature and duration, a method that preserved the scaffolds’ structure while ensuring thorough cleaning was developed. Scaffolds cleaned with LithaSol 80 had smoother surfaces and better pore structure preservation compared to those treated with DBE, which was more aggressive and caused some damage. This research represents a significant advancement in bone scaffold fabrication. By overcoming the cleaning challenges, the project has paved the way for producing personalized scaffolds tailored to real clinical cases. These optimized scaffolds are now ready for further biological and mechanical testing to ensure they meet the specific needs of bone tissue regeneration.
10. www.marketdataforecast.com/market-reports/europe-dental-bone-graft-substitutes-market
11. Xie et al., Titanium mesh for bone augmentation in oral implantology: current application and progress, International Journal of Oral Science 12 (2020) 37. https://doi.org/10.1038/s41368-020-00107-z
The AffordBoneS project focuses on creating affordable and customizable scaffolds for bone augmentation procedures to be used before dental implants placement. These scaffolds could also be used for other bone regeneration applications, expanding their potential market. Significant progress was made in the AffordBoneS project using ceramic vat photopolymerization to create complex bone scaffolds. These scaffolds closely mimic the structure of natural spongy bone, with pore sizes ranging from 10 to 900 micrometers and a total porosity of about 76%, making them ideal for bone regeneration. However, cleaning the uncured ceramic material from these intricate structures was challenging. To address this, various cleaning methods were tested. Traditional spray cleaning was ineffective, so alternative methods using soaking and ultrasonic cleaning with dibasic ester (DBE) and LithaSol 80 were explored. By optimizing the cleaning temperature and duration, a method that preserved the scaffolds’ structure while ensuring thorough cleaning was developed. Scaffolds cleaned with LithaSol 80 had smoother surfaces and better pore structure preservation compared to those treated with DBE, which was more aggressive and caused some damage. This research represents a significant advancement in bone scaffold fabrication. By overcoming the cleaning challenges, the project has paved the way for producing personalized scaffolds tailored to real clinical cases. These optimized scaffolds are now ready for further biological and mechanical testing to ensure they meet the specific needs of bone tissue regeneration.
10. www.marketdataforecast.com/market-reports/europe-dental-bone-graft-substitutes-market
11. Xie et al., Titanium mesh for bone augmentation in oral implantology: current application and progress, International Journal of Oral Science 12 (2020) 37. https://doi.org/10.1038/s41368-020-00107-z