Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Summary

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Abstract

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Introduction

Autologous bone is still the gold standard for repairing bone defects due to trauma, congenital defects, and surgical resection. However, autogenous bone grafting has significant limitations, including donor pain, risk of infection, and limited bone volume that can be isolated from the patients^1,2,3,4. Numerous biomaterials have been developed as bone substitutes, combining natural or synthetic polymers with mineralized materials such as calcium phosphate or hydroxyapatite^5,6. Bone f....

Protocol

This protocol uses 4-week-old male nude mice. House four mice in a cage under a 12 h light/dark cycle at 22−24 °C and 50%−70% relative humidity. All animal experiments were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (approval ID: A2019-204C, A2020-116A, and A2021-121A).

1. Preparation of buffers and reagents

1. Prepare mesenchymal stem cell growth mediu.......

Discussion

Using appropriate scaffold materials that promote the transition from hypertrophic cartilage to bone is a promising approach to scale up MSC-based engineered hypertrophic cartilage grafts and treat bone defects of clinically significant size. Here, we show that HA is an excellent scaffold material to support the differentiation of MSC-based hypertrophic cartilage tissue in vitro and to promote endochondral bone formation in vivo³⁸. Furthermore, in vivo, HA constructs wer.......

Materials

Name	Company	Catalog Number	Comments
0.25w/v% Trypsin-1mmol/L EDTA.4Na Solution	FUJIFILM Wako Pure Chemical	209-16941
Antisedan	Nippon Zenyaku Kogyo
ascorbate-2-phosphate	Nacalai Tesque	13571-14
Bambanker	GC Lymphotec	CS-02-001
basic fibroblastic growth factor	Reprocell	RCHEOT002
bovine serum albumin	FUJIFILM Wako Pure Chemical	012-23881	7.5 w/v%
Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4%	Invitrogen	C10283
dexamethasone	Merck	D8893
Domitor	Nippon Zenyaku Kogyo
Dormicum	Astellas Pharma
Dulbecco's Modified Eagle Medium	Merck	D6429	high glucose
Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham	Merck	D6421
Fetal bovine serum	Hyclone	SH30396.03
Gentamicin sulfate	FUJIFILM Wako Pure Chemical	1676045	10 mg/mL
Haccpper Generator	TechnoMax	CH-400-5QB	50 ppm hypochlorous acid water
Human Mesenchymal Stem Cells	Lonza	PT-2501
HyStem Cell Culture Scaffold Kit	Merck	HYS020
IL-1ß	PeproTech	AF-200-01B
ITS-G supplement	FUJIFILM Wako Pure Chemical	090-06741	×100
L-Alanyl-L-Glutamine	FUJIFILM Wako Pure Chemical	016-21841	200mmol/L (×100)
L-proline	Nacalai Tesque	29001-42
L-Thyroxine	Merck	T1775
MSCGM Mesenchymal Stem Cell Growth Medium BulletKit	Lonza	PT-3001
paraffin	FUJIFILM Wako Pure Chemical	165-13375
PBS / pH7.4 100ml	Medicago	09-2051-100
TGF-β3	Proteintech	HZ-1090
Vetorphale	Meiji Seika Kaisha
Visiocare Ointment	SAVAVET/SAVA Healthcare
β-glycerophosphate	FUJIFILM Wako Pure Chemical	048-34332