Recently, the latest research results of Biosis Healing SIS hydrogel "Microsphere-Gel Composite System with Mesenchymal Stem Cell Recruitment, Antibacterial, and Immunomodulatory Properties Promote Bone Regeneration via Sequential Release of LL37 and W9 Peptides", was published by the top journal in the field of applied materials "ACS Applied Materials & Interfaces", with an impact factor of 10.383.
This research was completed by the team of Professor Zhao Bo, Dr. Wei Pengfei from Biosis Healing, and the team of Professor Wang Yonglan from Tianjin Medical University, Doctor Liu Zihao from Tianjin Zhongnuo Stomatological Hospital, and the team of Dr. Ma Shiqing from Second Hospital of Tianjin Medical University. The main research object is the SIS hydrogel with independent intellectual property rights of Biosis Healing.
Currently, various types of biomaterials have been widely used clinically to treat complex bone defects. However, potential infection risks and inappropriate host immune responses posed by biomaterials may adversely affect the final bone repair effect. Therefore, the development of novel bone biomaterials with antibacterial and immunomodulatory capabilities can help to achieve a favorable interaction between the host and the material, thereby creating a microenvironment that is locally favorable for osteogenesis and ultimately accelerates bone regeneration.
In this study, we prepared LL37 peptide-containing SIS hydrogels and PLGA microspheres encapsulated with W9 peptide-- W9/PLGA-SIS, which can filled irregular bone defects and exhibited excellent mechanical properties.
We elaborate on the content of the article from several aspects.
Figure: Schematic diagram of preparation methods of SIS and LL37-W9/PLGA-SIS gels
1. The characterization of the hydrogel
The researchers first performed a series of characterizations on the synthesized samples, including SEM, FTIR, particle size distribution, fluorescence images, rheology, stress-strain, and peptide release profiles. The test results showed that although there was a certain degree of intermolecular interaction between PLGA microspheres and SIS collagen, it did not cause significant changes in the collagen structure, which also indicated the successful preparation of the microsphere-gel composite system. At the same time, the hydrogel has basic conditions suitable for the normal growth and proliferation of cells, and at the same time, the hydrogel can realize the sequential release of peptides, which is more conducive to the repair of bone defects.
Figure: Characterization of samples
2. Cytotoxicity Studies and Cell Cytoskeleton Observation
The researchers used BMSCs and RAW264.7 as model cells for cell culture, and the experiments showed that the hydrogel has excellent biocompatibility. The morphology and spreading of BMSCs and RAW264.7 cells were observed by CLSM after culturing with the sample extract for 1 day. The images illustrated that the cells had larger spreading areas and good stretching of actin filaments, suggesting that the LL37 peptide was more conducive to cell movement and cytoskeleton development.
Transwell assays were used to investigate the ability of the samples to promote BMSC recruitment. The LL37- W9/PLGA-SIS group greatly promoted the migration of BMSCs in vitro, which may be beneficial to the accumulation of BMSCs at the site of injury.
Figure: Effects of samples on cell viability, morphology, and behavior
The antibacterial properties of the samples were evaluated by the total antibacterial activity test, inhibition zone test, inactivity efficiency test, and live/dead staining. The number of bacterial colonies in the LL37-W9/PLGA-SIS group were significantly reduced, and only sporadic bacterial colonies could be seen. Quantitative analysis and fluorescence staining showed that it had good antibacterial properties.
Figure: Evaluation of antibacterial properties of samples
4. Effect of hydrogel on osteogenic differentiation in vitro
After clarifying the effect of macrophage factors on BMSC osteogenic differentiation mediated by materials, we further evaluated the expression of the typical osteogenesis-related genes RUNX2, OPN, BMP2, and ALP by qRT-PCR analysis. In addition, ALP and ARS staining vividly showed that the LL37-W9/PLGA-SIS group had the highest alkaline phosphatase expression and biomineralization. These results further demonstrate that factors secreted by macrophages cultured with LL37-W9/PLGA-SIS extracts can accelerate the material-mediated osteogenic differentiation of BMSCs.
Figure: Effect of BMSC CM on the process of macrophage polarization
5. Hydrogels used for defect repairing in vivo
We established 8 mm rat skull defect models and implanted gels to explore their osteogenic ability in situ. At 3 months after surgery, new bone formation increased in all groups, there were signs of fusion of bone islands and marginal bone in the LL37-W9/PLGA-SIS group. The quantitative bone volume fraction analysis (BV/TV) manifested that at 1 and 3 months after surgery, the LL37-W9/PLGA-SIS group had the highest rate of bone formation.
Figure: Hydrogels used for bone defect repairing in vivo
New bone formation and material degradation were further observed by H&E and Masson's trichrome staining. The results showed that the defects of the blank and SIS groups were mainly filled by loose fibrous connective tissues, with some local osteoid formation, while the LL37-SIS and LL37-W9/PLGA-SIS groups had new bone formation but no mature bone tissue. There were still undegraded microspheres in the LL37-W9/PLGA-SIS group, and new bone formation was observed around the microspheres, which may be due to the release of the W9 peptide during the degradation of PLGA microspheres.
Figure: Histological evaluation of bone regeneration
Immunohistochemical staining was used to further clarify the relationship between macrophage polarization and bone regeneration in vivo. Along with the gradual formation and maturation of new bone, the expression of BMP2 was decreased, while the expression of COL-1 was slightly increased. The expression levels of iNOS and CD206 returned to a relatively low and balanced state, which may indicate that subsequent tissue repair and reconstruction will be carried out in a relatively stable microenvironment. The microspheres were initially encapsulated by SIS gel and did not cause changes in the pH value of the surrounding environment or the inflammatory response. The degradation time of PLGA microspheres was longer than that of the SIS gel, which may cause the up-regulated expression of iNOS in a limited range, but this did not cause a severe inflammatory response or impair the final osteogenic effect.
Figure: Immunohistochemical staining were used to assess bone formation in vivo
In this study, we successfully prepared SIS hydrogel containing LL37 peptide and PLGA microspheres encapsulated with the W9 peptide, which have good biocompatibility and mechanical properties. In addition, this microsphere−gel composite system can sequentially release LL37 and W9 peptides. Verified by in vitro studies, it was found that this system not only has good BMSC recruitment ability and antibacterial properties but also precisely regulates the polarization process of macrophages and promotes the osteogenic differentiation of BMSCs in a way that is closer to physiological bone healing. The biocompatibility, cell recruitment ability, cell recruitment ability and bone formation ability of LL37-W9/PLGA-SIS gel were verified by in vitro and in vivo experiments, which provided experimental basis for clinical treatment of bone defects.
Zhao Bo
Biosis Healing founder, doctor of surgery
Professor-level senior engineer/ associate chief physician/ graduate supervisor/ postdoctoral supervisor
As the main completion and project leader, he has undertaken and completed four projects of National Natural Science Foundation of China, Beijing Natural Science Foundation and many scientific research projects.
Wang Yonglan
Chief Physician, Director of the Teaching and Research Section of Oral Medicine, School of Stomatology, Tianjin Medical University, Director of the Department of Periodontics, Master Supervisor
Standing Committee Member of Periodontology Professional Committee of Chinese Stomatological Association
Chairman of the Periodontology Professional Committee of Tianjin Stomatological Association
Presided over and participated in more than 20 research projects at the bureau level, municipal level and national level, and published 40 papers
Won 1 third prize of Tianjin Science and Technology Achievement Award, 1 second prize of Tianjin Medical University Education Reform Award, and 4 blank prizes for introduction and application of new technologies in Tianjin health system Participated in 4 books
Won the "Top Ten Doctors" and "Honorary Title of Women's Contribution Model" in the first Doctor's Day of Tianjin Medical University
The first "Good Doctor" that the people are satisfied with and "May 1st" Labor Medal Winner by Tianjin Municipal Health Bureau
Liu Zihao
Doctor of Stomatology, Attending Doctor, Lecturer
Youth committee of the Professional Committee of Stomatology Education of the
Chinese Stomatology Association
131 Third-level Talents in Tianjin
Award Tianjin Education Commission fund, Health Commission fund
Award The second prize of Tianjin Scientific and Technological Progress in 2020
Ma Shiqing
Department of Stomatology, Second Hospital of Tianjin Medical University, Lecturer, Attending Doctor
Young committee of the Professional Committee of General Oral of the Chinese Stomatological Association
Research direction is guided tissue regeneration and implant surface modification Presided over 1 youth project of the National Natural Science Foundation of China In the past 5 years, published 5 SCI papers as the first author and 1 SCI paper as the corresponding author
Won the second prize of Tianjin Science and Technology Progress Award
131 Third-level Talents in Tianjin
Candidates for the "Young Reserve Talents Support Program" in Tianjin Universities
Outstanding Teacher of Tianjin Medical University
Wei Pengfei
Beijing Biosis Healing Biotechnology Co., Ltd. Post-doctoral
Doctor of Biomedical Materials, Beijing University of Chemical Technology
He was supported by the 68th batch of China Postdoctoral Science Foundation
2020 Annual Postdoctoral International Exchange Program Academic Exchange Project
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