Revolutionizing bone regeneration and wound healing: Mechanical stromal vascular fraction and hyaluronic acid in a mouse calvarial defect model

Abstract

Introduction: The stromal vascular fraction (SVF) is a complex and heterogeneous suspension derived from adipose tissue, containing both cellular and noncellular components. Its cellular fraction includes adipose-derived stem cells (ASCs), endothelial precursor cells, pericytes, macrophages, lymphocytes, and smooth muscle cells. The acellular "secretome" of SVF includes bioactive molecules such as growth factors, cytokines, chemokines, extracellular vesicles, and fragments of extracellular matrix (ECM), which contribute to its regenerative potential. Bone defeatures can be stimulated by mesenchymal stem cells (MSCs) that differentiate into osteoblast to support the healing and repair process. In addition to its cell content, the SVF is rich in growth factors, cytokines and chemokines, extracellular vesicles, and extracellular matrix components, which could stimulate regenerative processes through a trophic effect. Studies showed that hyaluronic acids are usually involved in healing processes. This study was focused on the healing potency of stromal stem cells isolated from adipose tissues by mechanical digestion, and the role of low-molecular-weight hyaluronic acid (LMW-HA, ACP) in the healing process was tested in calvarial defeatures in a mouse model, in comparison with the enzymatic digestion method.

Methods: The bone healing and remodeling process was evaluated in vivo using magnetic resonance imaging (MRI) up to 15 days post-treatment, and differences in the quality of bone regeneration were assessed by ex vivo histological analysis, immunofluorescences, and ultrastructural analysis. The bone matrix formed after treatment with mechanically digested Hy tissue stromal vascular fraction + hyaluronic acid (HT-SVF + ACP) was compared to that formed with enzymatically digested stromal vascular fraction + hyaluronic acid (ED-SVF + ACP), with the saline group serving as the control group.

Results: In this study, we explore a groundbreaking approach using HT-SVF combined with ACP to promote bone regeneration. Through comparative analysis with ED-SVF in a calvarial defect mouse model, we demonstrate the superior efficacy of HT-SVF + ACP in enhancing bone healing, reducing fibrotic tissue, and improving bone matrix maturity.

Discussion: The findings establish the potential of HT-SVF as a cost-effective and efficient method for bone regenerative therapy.

Keywords: adipose-derived stem cell; bone repair; calvarial bone defects; hyaluronic acid; stromal vascular fraction.

FIGURE 1

The cell viability and population of ED-SVF and HT-SVF were characterized by flow cytometry by using the P.I., CD34, CD105, and CD73 markers. Data are expressed in percentage of positive cells ±SEM.

FIGURE 2

Visual observation of the wound after 15 days post-surgery. The black arrows indicate the wounds. All the experimental groups are presented, including controls (saline and ACP) and treatments (HT-SVF and ED-SVF).

FIGURE 3

Representative MRI of the wound healing process at 5 and 15 days after the operation. The squares are representative for the magnifications, and the white arrows indicate the injury sites. All the experimental groups are presented, including controls (saline and ACP) and treatments (HT-SVF and ED-SVF).

FIGURE 4

Morphological evaluation with hematoxylin/eosin staining. The squares represent the magnifications in the right column, the black lines represent the initial hole diameter, and the dotted lines represent the newly formed bone area and scar tissue. All the experimental groups are presented, including controls (saline and ACP) and treatments (HT-SVF and ED-SVF).

FIGURE 5

Glycosaminoglycan evaluation with Alcian blue/Fast red staining. The squares represent the magnifications in the right column, the black lines represent the initial hole diameter, and the dotted lines represent the newly formed bone area. All the experimental groups are presented, including controls (saline and ACP) and treatments (HT-SVF and ED-SVF).

FIGURE 6

Calcium deposit evaluation with Alizarin red and hematoxylin. The squares represent the magnifications in the right column, the black lines represent the initial hole diameter, and the dotted lines represent the magnification zone of the newly formed area. All the experimental groups are presented, including controls (saline and ACP) and treatments (HT-SVF and ED-SVF).

FIGURE 7

Immunofluorescence analysis of HLA⁺ human cells. The columns consist of different experimental groups (CTRL+, saline, ACP, ED-SVF + ACP, and HT-SVF + ACP). Each row represents different markers, including nuclei marker (DAPI, blue), human leukocyte antigen marker (HLA, green), and composite of markers. All the images were acquired with a ×20 magnification objective.

FIGURE 8

Immunofluorescence analysis of the newly formed bone marker. The columns consist of different experimental groups (CTRL+, saline, ACP, ED-SVF + ACP, and HT-SVF + ACP). Each row represents different markers, including nuclei marker (DAPI, blue), bone neoformation marker (OPN, green), and composite of markers. All the images were acquired with a ×20 magnification objective.

FIGURE 9

Ultrastructural analysis using scanning electron microscopy (SEM). The squares represent the magnification areas, and the right column represents its further magnification. All the experimental groups are presented, including controls (saline and ACP) and treatments (HT-SVF and ED-SVF).