Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Published: May 31, 2024

doi:

Tianqi Li^2,3, Xiangbo Meng², Shuaichen Li², Sunxin Zhou², Hongkun Li², Shi Quan, Tong Zhang

¹Medical School of Chinese PLA, ²Department of Stomatology, the First Medical Centre,Chinese PLA General Hospital, ³Department of Stomatology,Fujian People’s Armed Police Corps Hospital

Summary

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Abstract

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a 'niche-based approach on stemness' for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a 'fibroblast-like' spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Introduction

Mesenchymal stem cells (MSCs) were first discovered in bone marrow, which showed the ability to form adhesive colonies in culture and strong osteogenic potential¹. Pittinger et al.² further found their multi-directional differentiation potential towards bone, fat, and cartilage. Although all mesenchymal stem cells from different sources have the potential for multi-directional differentiation, bone marrow mesenchymal stem cells have the strongest chondrogenic differentiation potential compared with mesenchymal stem cells derived from other tissues, making them considered the best candidate cells for bone tissue engineering³. However, many studies have proved that BMMSCs from different origins present site-specific characteristics and properties such as osteogenic differentiation capability and cellular proliferative activity⁴^,⁵. This may be due to different germ layers between the jaw and appendicular bones or the iliac crest⁶.

Jaw bone marrow mesenchymal stem cells (JBMMSCs) arise from neural crest cells of the neuroectoderm, while femur-derived BMMSCs originate from the mesoderm⁷. Compared with BMMSCs derived from long bones and the iliac crest, JBMMSCs have a higher proliferation rate, ALP activity, and osteogenic potential⁸. Besides, the application effect of BMMSCs in tissues and organs may vary depending on different cell types and environments⁹. Repair of jaw defects mainly depends on the recruitment of jaw-derived mesenchymal stem cells. Therefore, studying JBMMSCs can provide an experimental basis for its clinical application in jaw bone tissue engineering¹⁰. However, basic research and clinical applications mainly focus on BMMSCs derived from appendicular and axial bones¹¹. Research on JBMMSCs is limited, and this may be due to the low content of cancellous bone in the mandible and the fact that rats’ jaws have even less cancellous bone content¹². Therefore, it is difficult to separate jaw bone-derived marrow mesenchymal stem cells using the ordinary bone marrow flushing method, which is commonly used in isolating BMMSCs from appendicular bones or the iliac crest¹³. Based on the methods of Hong et al.¹⁴ and Cheng et al.¹⁵, we hypothesized that the combination of dense bone digestion and the bone marrow flushing method could efficiently isolate rat JBMMSCs.

This study aims to establish an efficient method for isolating rat JBMMSCs and provide sufficient seed cell sources for jaw bone tissue engineering.

Protocol

The protocol was approved by the Institutional Animal Ethics Committee of the Chinese PLA General Hospital. Thirteen-week-old male Wistar rats were used for the experiment. Details on the animals, reagents, and equipment are listed in the Table of Materials. 1. Experimental preparation Sterilize all surgical instruments, including ophthalmic scissors, tweezers, and bone rongeurs, at high temperature and pressure. Prepare culture media in …

Representative Results

After 72 h of cell inoculation, most cells were suspended and round in shape, with very few adhered to the wall (Figure 1B). By the fifth day, adherent cell colonies appeared, exhibiting spindles or fibroblast-like shapes (Figure 1C). By the seventh day, adherent cells reached 90% confluency, forming a "fish school" shape with a small number of intermittent suspended cells (Figure 1D). Passaged cells grew rapidly and were pa…

Discussion

Bone marrow mesenchymal stem cells (BMMSCs) represent a subset of non-hematopoietic stem cells residing in bone marrow, characterized by their self-renewal capabilities, multi-directional differentiation potential, and supportive functions for hematopoiesis. These cells play pivotal roles in various physiological processes such as tissue regeneration, angiogenesis, and the regulation of cellular activities²⁰. Consequently, BMMSCs are frequently utilized in tissue repair and regenerative engineerin…

Divulgations

The authors have nothing to disclose.

Acknowledgements

This study was supported by the Health Care Projects of the Military Commission Logistics Department (19BJZ22), Beijing Natural Science Foundation (7232154), and Fourth Mil Med Univ. clinical research projects (2021XB025).

Materials

Alizarin Red S Solution 0.2%	Solarbio	G1450
BCIP/NBT Alkaline Phosphatase Color Development Kit	Beyotime	C3206
Bio-Rad CFX96 Real-Time System	Bio-Rad
CCK8 Kit	Dujindo	CK04
Cell culture dish 10 cm	Corning	353003
Centrifuge	Eppendorf	5810R
Centrifuge Tube 15 mL	Corning	430790
Centrifuge Tube 50 mL	Corning	430828
CO₂ incubator	Thermo Fisher	3111
Constant-temperature oscillator	Shanghai Zhicheng Analysis Instrument Manufacturing Co., Ltd.	ZWY-100H
Fetal bovine serum	BI	04-001-1ACS
Flow cytometer	BD	FACS C6
Inverted phase-contrast microscope	Olympus	CKX41
Mesenchymal Stem Cell (Rat) Surface marker Detection Kit	Oricell	RAXMX-09011
Multifunctional microplate reader	BioTek	Synergy LX Multi-Mode
Oil Red O Stain Kit	Solarbio	G1262
Paraformaldehyde 4%	Solarbio	P1110
PBS	MACGENE	CC008
penicillin-streptomycin 0.25%	MACGENE	CC004
PowerUp SYBR Green Master Mix	Thermo Fisher	A25742
PrimeScript RT Master Mix	Takara	RR036A
Rat Bone Marrow Mesenchymal Stem Cells Adipogenic Differentiation kit	Oricell	RAXMX-90031
Rat Bone Marrow Mesenchymal Stem Cells Chondrogenic Differentiation kit	Oricell	RAXMX-90041
Rat Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation kit	Oricell	RAXMD-90021
RNA extraction kit	TIANGEN	DP419
Super-clean bench	Beijing Yataikelong Instrument Technology Co. Ltd.	KLCZ-1220A
Trypsin-EDTA 0.25%	MACGENE	CC012
Type II collagenase	Solarbio	C8150
Wistar rat	Beijing Yataikelong Instrument Technology Co. Ltd.
α-MEM culture medium	Gibco	C12571500BT

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