Incorporating Bone-Derived ECM into Macroporous Microribbon Scaffolds to Accelerate Bone Regeneration

Orthopedics Immunology Life Sciences Cell Biology Bioengineering Medicine
bone regeneration macroporous scaffolds extracellular matrix tissue engineering immunomodulation

Incorporating Bone-Derived Extracellular Matrix (Bone-Derived ECM) into Macroporous Microribbon Scaffolds Accelerates Bone Regeneration Research Report

With the continuous progress in the biomedical field, the application of tissue engineering and regenerative medicine in various tissue repairs has become increasingly important. However, research on bone tissue regeneration still faces numerous challenges. Bone injuries and insufficient regenerative capacity are pressing issues in modern medicine, especially in cases where aging and specific diseases significantly reduce bone regeneration capacity (e.g., bone defects and trauma). Critical-Sized Bone Defects are a type of bone injury that cannot heal on its own. Current treatment methods include autologous or allogeneic bone grafts. However, these treatments are limited by donor tissue availability, donor site morbidity, and immunogenicity. Therefore, developing innovative materials to mimic the bone matrix environment and promote bone regeneration has become a critical scientific issue.

Against this backdrop, this study focuses on developing macroporous microribbon scaffolds (Macroporous Microribbon Scaffolds, referred to as μRB) combined with bone-derived extracellular matrix (Bone-Derived ECM, referred to as BECM) to accelerate bone tissue regeneration. Conducted by Cassandra Villicana, Ni Su, Andrew Yang, and others from Stanford University School of Medicine, the study was published in the journal Advanced Healthcare Materials. The research demonstrates that integrating BECM with macroporous scaffolds significantly enhances bone tissue repair and promotes vascularization.

Research Overview and Objectives

Background and Problem Statement

Tissue-Derived Extracellular Matrix (TDECM) is a widely used biomaterial with highly biomimetic properties that can induce regenerative immune responses in vivo. It is currently extensively applied in soft tissue regeneration. However, existing TDECM materials are mostly nanoporous structures (Nanoporous Structure), and their permeability and cell infiltration limit their potential for hard tissue (e.g., bone tissue) regeneration. In contrast, Macroporosity is crucial for accelerating cell infiltration and bone formation. Yet, there is a lack of strategies to combine macroporosity with ECM-based hydrogels, particularly for bone repair applications. Additionally, the performance of pure BECM materials is insufficient to support efficient bone regeneration.

Therefore, this study aims to integrate bone-derived extracellular matrix with gelatin-based microribbon scaffolds using a novel Co-Spinning Technique and optimize their material composition to achieve more efficient bone regeneration.

Research Process

The study was conducted in the following steps:

1. Isolation and Characterization of BECM

The study began by processing cancellous bone from bovine tibiae using established decellularization protocols, including demineralization, lipid extraction, and decellularization, to obtain decellularized bone matrix (BECM).
- Key Confirmed Components: The residual cell nuclei were quantified using the Picogreen DNA Assay, followed by hematoxylin and eosin (H&E) staining. The main protein components of BECM were collagen, along with other non-collagen proteins and extracellular matrix regulators.
- Mass spectrometry (Mass Spectrometry) analysis confirmed that the major biological functional proteins in BECM could enhance endochondral bone morphogenesis, giving it significant potential for bone regeneration.

2. Integration of BECM with μRB Scaffolds and Optimization of Properties

Using an adapted co-spinning method based on wet spinning, BECM at varying weight ratios (0%, 15%, 25%, 50%) was mixed with methacrylated gelatin (GelMA) solution to form microribbon structures, which were then assembled into three-dimensional macroporous scaffolds.
- Scaffold Morphology and BECM Distribution: Confocal imaging and scanning electron microscopy (SEM) revealed uniform BECM distribution, with the best integration observed in the 15%-25% BECM groups. Porosity quantification showed that the macroporosity of the scaffolds remained unaffected (≈20% porosity) when BECM was added at up to 25%.
- Mechanical and Physical Property Testing: Further mechanical analysis indicated that BECM at ratios below 25% did not significantly alter the scaffold’s modulus (Stiffness). However, at 50%, the modulus decreased significantly, reducing scaffold stability.

3. In Vitro Performance Testing: Analysis of MSC Osteogenic Potential

To validate the osteogenic promotion effect of BECM, mouse mesenchymal stem cells (MSCs) were encapsulated in the scaffolds and cultured for up to 4 weeks.
- Early and Late Osteogenic Markers: Immunofluorescence labeling of the early osteogenic marker RUNX2 showed that the 15% BECM group significantly upregulated its expression. Alizarin Red S (ARS) staining also confirmed that the 15% group exhibited superior mineralization compared to other groups.
- Increased Osteoblast Matrix Production: Masson’s Trichrome staining indicated that the 15% BECM scaffold demonstrated the best promotion of extracellular matrix deposition and osteoblast generation.

4. Enhancing In Vitro Osteogenesis: Addition of Tricalcium Phosphate (TCP) Particles

The study further enhanced the regulatory effect by adding a small amount (0.5%) of TCP particles to the scaffolds. In vitro data showed that scaffolds combined with TCP significantly improved the expression of MSC-RUNX2 and the mature bone protein Osteocalcin (OCN), with the 15% BECM group remaining the best performer.

5. In Vivo Validation: Endogenous Repair in a Mouse Cranial Bone Defect Model

Using a Critical-Sized Cranial Bone Defect Model in mice, the study systematically evaluated the osteogenic promotion effect of BECM-μRB scaffolds in endogenous bone regeneration. MicroCT imaging revealed that the 15% BECM group combined with TCP particles effectively filled the defect (≈55%) by week 2 and significantly increased the bone mineral density (BMD) of the newly formed bone. Additionally, the 15% group exhibited the highest level of vascularization (CD31 Immunostaining).

6. Immunomodulation and Macrophage Phenotype Effects

The study explored the regulatory effect of BECM on macrophage (M𝜓ϕ) immune responses. One week after scaffold implantation, the 15%-25% BECM groups significantly reduced the proportion of pro-inflammatory M1 phenotype (CD86+) and contributed to a more regenerative immune environment for bone tissue repair.

Research Results and Findings

  1. A novel macroporous biomaterial combining BECM with GelMA microribbon scaffolds was successfully developed. Optimizing the BECM content in the scaffolds (between 15%-25%) significantly enhanced their mechanical, biological, and bone regeneration properties.
  2. The synergistic application of TCP particles further improved the osteogenic effect of BECM scaffolds, demonstrating the necessity of mineralization cues in bone regeneration strategies.
  3. In terms of the immune environment, BECM scaffolds modulated macrophage phenotype responses, creating a more favorable environment for bone regeneration.

Significance and Contributions

  1. This study provides an innovative and efficient platform for hard tissue regeneration, particularly suitable for Critical-Sized Bone Defects.
  2. The highly modular nature of microribbon scaffolds allows for the integration of other types of TDECM into similar constructs, enabling future applications in cartilage, muscle, and other tissue regeneration fields.
  3. The study demonstrates that significant bone regeneration can be achieved even without exogenous cells or growth factors by combining BECM with TCP particles, reducing the complexity and cost of clinical applications.

Highlights

  1. The first application of a pioneering BECM co-spinning technique in macroporous scaffolds.
  2. Confirmation of the new potential of Minimally Invasive Scaffolds (μRB) combined with multifactorial stimulation in bone repair.
  3. New insights into the regulation of the bone immune microenvironment and endogenous tissue healing mechanisms.

This study holds high practical value and scientific significance in the field of bone tissue engineering and provides an important reference for the development of materials for various tissue regeneration applications in the future.