Regenerative performance and immunogenicity of engineered extracellular matrices.
(2025) In Lund University, Faculty of Medicine Doctoral Dissertation Series- Abstract
- Critical-sized bone defects remain a significant unmet clinical need, with > 500,000 reconstructive procedures performed annually in the United States and Europe and associated costs exceeding US$3 billion. Autologous bone grafting—the current gold standard—suffers from limited harvest volume and donor-site morbidity, whereas allografts show variable integration and carry immunological and infection risks. Recombinant therapies using growth factors partly address these issues but require supraphysiological doses that provoke ectopic ossification and inflammation. Tissue engineering (TE) offers a conceptual solution by combining cells, bioactive cues, and scaffolds to fabricate living grafts; however, cell-laden constructs that rely on... (More)
- Critical-sized bone defects remain a significant unmet clinical need, with > 500,000 reconstructive procedures performed annually in the United States and Europe and associated costs exceeding US$3 billion. Autologous bone grafting—the current gold standard—suffers from limited harvest volume and donor-site morbidity, whereas allografts show variable integration and carry immunological and infection risks. Recombinant therapies using growth factors partly address these issues but require supraphysiological doses that provoke ectopic ossification and inflammation. Tissue engineering (TE) offers a conceptual solution by combining cells, bioactive cues, and scaffolds to fabricate living grafts; however, cell-laden constructs that rely on the in vitro expansion and differentiation of primary cells face considerable challenges, including variability between donors, extended production times, reduced viability upon implantation, and limited scalability. Engineered extracellular matrices (ECMs), acellular scaffolds deposited by cells and subsequently devitalized, offer a way to overcome these barriers by preserving intrinsic biological cues, thereby addressing the logistical and safety challenges associated with cell-based therapies. This thesis explores how eECMs can be optimized for bone regeneration by addressing four critical translational barriers: growth factor dependency, cell donor variability, programmable matrix function, and immune compatibility.
First, we show that a collagen–hydroxyapatite scaffold loaded with heterodimeric Bone morphogenetic protein (BMP) 2/7 significantly improves osteoinduction at lower doses than BMP-2 alone, enhancing osteogenic progenitor recruitment and matrix deposition (Paper 1). We then demonstrate the development of an off-the-shelf eECM generated from a standardized human mesenchymal stem/stromal cell line (MSOD-B), which, upon devitalization, supports robust endochondral ossification in a cell-free manner (Paper 2). We applied CRISPR/Cas9 editing to customize the composition and function of eECMs by targeting key regulators of endochondral ossification. Vascular endothelial growth factor (VEGF) knockout in MSOD-B cells yielded cartilage matrices that, despite delayed vascularization, retained full capacity to prime endochondral ossification in ectopic models, indicating that VEGF is dispensable for initiating this program. In contrast, Runt-related transcription factor 2 (RUNX2) knockout prevented cartilage hypertrophy and significantly reduced ossification, yet enhanced cartilage repair in a rat osteochondral defect model. These findings demonstrate that transcriptional engineering of ECM-producing cells enables the precise modulation of regenerative outcomes, allowing for programmable shifts between osteogenic and chondrogenic pathways (Paper 3). Finally, we characterized the immune response to ECMs from cartilage (Paper 4) and 3D-printed lung tissues (Paper 5) in various animal models, highlighting the correlation between early M2 macrophage recruitment and tissue regeneration, and noting the variability in the predictive outcomes of tissue regeneration based on early immune recruitment patterns.
In summary, we demonstrated the performance of ECMs in instructing tissue repair using standardized cell lines, of which genetic customization leads to tailored graft properties. We determined the immunogenicity of ECMs and revealed early immune response patterns in engineered and 3D-printed ECMs associated with successful tissue regeneration. Our research contributes to the development of effective repair strategies beyond the skeletal framework, facilitating the advancement of next-generation, personalized grafts.
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Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/992f8684-243c-49b2-b802-af6bf5be10b7
- author
- Prithiviraj, Sujeethkumar LU
- supervisor
-
- Paul Bourgine LU
- Filipe Pereira LU
- opponent
-
- Associae Professor Papantoniou, Ioannis, KU Leuven, Belgium
- organization
- publishing date
- 2025
- type
- Thesis
- publication status
- published
- subject
- keywords
- Engineered Extracellular Matrices, Customizable matrices, CRISPR Cas 9, Decellularization
- in
- Lund University, Faculty of Medicine Doctoral Dissertation Series
- issue
- 2025:107
- pages
- 98 pages
- publisher
- Lund University, Faculty of Medicine
- defense location
- GK-Salen, Forum Medicum, BMC I1146, Sölvegatan 19, Lund. Join by Zoom: https://lu-se.zoom.us/j/69666370158
- defense date
- 2025-10-20 13:00:00
- ISSN
- 1652-8220
- ISBN
- 2025: 978-91-8021-760-6
- language
- English
- LU publication?
- yes
- id
- 992f8684-243c-49b2-b802-af6bf5be10b7
- date added to LUP
- 2025-09-26 13:36:04
- date last changed
- 2025-09-30 07:32:20
@phdthesis{992f8684-243c-49b2-b802-af6bf5be10b7,
abstract = {{Critical-sized bone defects remain a significant unmet clinical need, with > 500,000 reconstructive procedures performed annually in the United States and Europe and associated costs exceeding US$3 billion. Autologous bone grafting—the current gold standard—suffers from limited harvest volume and donor-site morbidity, whereas allografts show variable integration and carry immunological and infection risks. Recombinant therapies using growth factors partly address these issues but require supraphysiological doses that provoke ectopic ossification and inflammation. Tissue engineering (TE) offers a conceptual solution by combining cells, bioactive cues, and scaffolds to fabricate living grafts; however, cell-laden constructs that rely on the in vitro expansion and differentiation of primary cells face considerable challenges, including variability between donors, extended production times, reduced viability upon implantation, and limited scalability. Engineered extracellular matrices (ECMs), acellular scaffolds deposited by cells and subsequently devitalized, offer a way to overcome these barriers by preserving intrinsic biological cues, thereby addressing the logistical and safety challenges associated with cell-based therapies. This thesis explores how eECMs can be optimized for bone regeneration by addressing four critical translational barriers: growth factor dependency, cell donor variability, programmable matrix function, and immune compatibility. <br/>First, we show that a collagen–hydroxyapatite scaffold loaded with heterodimeric Bone morphogenetic protein (BMP) 2/7 significantly improves osteoinduction at lower doses than BMP-2 alone, enhancing osteogenic progenitor recruitment and matrix deposition (Paper 1). We then demonstrate the development of an off-the-shelf eECM generated from a standardized human mesenchymal stem/stromal cell line (MSOD-B), which, upon devitalization, supports robust endochondral ossification in a cell-free manner (Paper 2). We applied CRISPR/Cas9 editing to customize the composition and function of eECMs by targeting key regulators of endochondral ossification. Vascular endothelial growth factor (VEGF) knockout in MSOD-B cells yielded cartilage matrices that, despite delayed vascularization, retained full capacity to prime endochondral ossification in ectopic models, indicating that VEGF is dispensable for initiating this program. In contrast, Runt-related transcription factor 2 (RUNX2) knockout prevented cartilage hypertrophy and significantly reduced ossification, yet enhanced cartilage repair in a rat osteochondral defect model. These findings demonstrate that transcriptional engineering of ECM-producing cells enables the precise modulation of regenerative outcomes, allowing for programmable shifts between osteogenic and chondrogenic pathways (Paper 3). Finally, we characterized the immune response to ECMs from cartilage (Paper 4) and 3D-printed lung tissues (Paper 5) in various animal models, highlighting the correlation between early M2 macrophage recruitment and tissue regeneration, and noting the variability in the predictive outcomes of tissue regeneration based on early immune recruitment patterns.<br/>In summary, we demonstrated the performance of ECMs in instructing tissue repair using standardized cell lines, of which genetic customization leads to tailored graft properties. We determined the immunogenicity of ECMs and revealed early immune response patterns in engineered and 3D-printed ECMs associated with successful tissue regeneration. Our research contributes to the development of effective repair strategies beyond the skeletal framework, facilitating the advancement of next-generation, personalized grafts.<br/>}},
author = {{Prithiviraj, Sujeethkumar}},
isbn = {{2025: 978-91-8021-760-6}},
issn = {{1652-8220}},
keywords = {{Engineered Extracellular Matrices; Customizable matrices; CRISPR Cas 9; Decellularization}},
language = {{eng}},
number = {{2025:107}},
publisher = {{Lund University, Faculty of Medicine}},
school = {{Lund University}},
series = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
title = {{Regenerative performance and immunogenicity of engineered extracellular matrices.}},
url = {{https://lup.lub.lu.se/search/files/228517707/2025-10-20_Prithiviraj_Final.pdf}},
year = {{2025}},
}