Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Summary

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Abstract

Osteoarthritis (OA) is one of the most common debilitating disorders. Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Introduction

Mesenchymal stem cells (MSCs) are known to have the capability to regenerate cartilage^1,2,3,4,5,6. They can be easily obtained from various sources: bone marrow, cord blood, and adipose tissue among many others. Among these sources, adipose tissue is the only source where a sufficient number of MSCs can be obtained without any culture expansion to regenerate cartilage in clinical settings^7,8. Autologous bone marrow....

Protocol

The approval and consent to report following case reports were waived by Myongji University Institutional Review Board committee (MJUIRB). Further, this clinical protocol was in compliance with the Declaration of Helsinki and regulation guidelines of the KFDA. For the procedures, informed consents were obtained from the patients.

1. Liposuction

NOTE: Perform with sterile technique.

1. Use the following inclusion criteria: (1) MRI evidence of stage 3 OA.......

Discussion

In 2001, Zuk et al. isolated stem cells from adipose tissue by breaking down the collagen matrix with collagenase⁶. Afterwards, the group showed that these stem cells isolated from the adipose tissue could transform into cartilage and other tissues of mesoderm in origin, proving that these stem cells were mesenchymal in origin. 

Likewise, the procedure presented in this article is a modified protocol to apply the similar method to human patients. The main .......

Materials

Name	Company	Catalog Number	Comments
Material
5% Betadine (povidone-iodine)	Firson Co., Ltd.	657400260
2% Lidocaine	Daehan Pharmaceutical Co.	670603480
Tumescent solution	Myungmoon Pharm. Co. Ltd.	N01BB01	The solution was composed of 500 mL normal saline, 40 mL 2% lidocaine, 20 mL 0.5% marcaine, and 0.5 mL epinephrine 1:1000.
Liberase TL and TM research grade	Roche Applied Science	5401020001
D5LR	Dahan Pharm. Co., Ltd.	645101072	Dextrose 5% in lactated Ringer's solution
Anticoagulant citrate dextrose solution	Fenwal, Inc.	NDC:0942-0641	The solution was composed of 0.8% citric acid, 0.22% sodium citrate, and 0.223% dextrose.
3% (w/v) Calcium chloride	Choongwae Pharmaceutical Co.	644902101
0.5% (w/v) HA (Hyaluronic acid )	Dongkwang pharm. Co., Ltd.	645902030
0.25% Ropivacaine	Huons Co., Ltd.	670600150
Equipment
3.0 mm Cannula	WOOJU Medical Instruments Co.	ML30200
60-mL Luer-Lock syringe	BD (Becton Dickinson)	309653
Centrifuge Barrel Kit	CPL Co., Ltd.	30-0827044
Tissue homogenizer that contains blades	CPL Co., Ltd.	30-0827045
Rotating incubator mixer	Medikan Co., Ltd	MS02060092
Centrifuge	Hanil Scientific Inc.	CE1133
Magnetic Resonance Imaging	Philips Medical Systems Inc.	18068
Ultrasound Imaging System	Samsung Medison co., Ltd	CT-LK-V10-ICM-09.05.2007