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Complications in Regenerative Medicine

Ricardo Ruiz-Lopez ¹, Yu Chuan Tsai MD ², and Mattia Squarcia MD³

¹,³ Clinica vertebra, Spine and Pain Surgery Centers, Barcelona / Madrid, Catalonia, Spain
² National Cheng Kung University, Tainan, Taiwan

Part I: Mesenchymal Cells Therapies

Introduction

Regenerative medicine (RM), including platelet-rich plasma therapy and stem-cell therapy involves delivery of specific types of cells or cell products to diseased tissues or organs, where they will ultimately restore tissue and organ function [1, 2].

RM has been applied to the treatment of pain as an optional and promising approach for the treatment of degenerative disc disease, osteoarthritis, neuropathic pain and musculoskeletal pain which does not respond to conventional medicine [3–7]. RM therapy utilizes the body’s innate capacity to heal itself using the body’s own biology to elicit a healing response.

Stem cells have the capacity of replicate and differentiate (Figure 55.1).

**Figure 55.1** MSCs can be derived from multiple tissue sources.

There are three types of stem cells; embryonic stem cells (ESC), induced pluripotent stem cells (iPS) and mesenchymal stem cells (MSC). ESC can differentiate in any tissue of our body. They are obtained in the inner cell mass of the blastocyst, a very early stage/embryo. ESC are also known as pluripontent stem cells [8].

iPS are stem cells that are obtained by human blood and are then genetically programmed to become a pluripotent stem cell and can then differentiate in any tissue cell of the human body [9].

MSC can be found everywhere in the human body and can differentiate in a variety of cells along the tissues. MSC are also defined as multipontent cells [10]. Physiologically, MSC replace damaged or used up cells and are essential for keeping the human body in a healthy state. MSC were first described in 1970 by Arnold Caplan who gave his name to stromal stem medicinal signaling cells or marrow signaling cells [11]. The name “mesenchymal stem cell” defines cells able to produce osteogenesis, condrogenesis adipogenesis with negative markers for CD34. Since the first Caplan definition of mesenchymal stromal cells, many different terms have been used when referring to MSC, for example, marrow stromal cells, multipontent stromal cells and mesodermal stem cells (Figure 55.2).

**Figure 55.2** MSC are derived from pericytes of blood vessels.

Stem cells are a cell-based therapy and have the ability to sense the environment and respond over weeks or even months. MSC are the most potent type of cells that we harvest from adult bodies. Mesenchymal stem cells for therapeutic injections for targeted regeneration of tissues can be harvested from two areas of the human body where they are very prevalent: bone marrow and adipose tissue [12]. The number of MSC found in adipose tissue is substantially higher in number than in bone marrow [13] (Figure 55.3).

**Figure 55.3** Stem cell division and differentiation.

When we pull a bone marrow or adipose tissue and concentrate the white blood cells, a small portion of these nucleated blood cells are MSC. When injected back into the damaged area, MSC can respond to inflammation by secreting a number of growth factors that modulate and control the levels of inflammation and to move from inflammation to remodeling and homeostasis [14].

Growth factors secreted by MSC can be anti-scarring signals (KGF, SDF-1, MIP 1a and 1b), anti-apoptosis (STC-1, SFRP2, TGF-beta1, VEGF and HGF), angiogenic (VEGF, TGF-beta 1), mitogenic (TGF-alfa, TGF-beta, IGF-1,FGF-2, EGF) anti-inflammatory (IL-6, IL-8, IL-10, PGE2, IDO, HGF, TSG-6 and NO). MSC finally returns to the vessel as pericytes. Different studies suggest that when using MSC in regenerative medicine, there is a dose-response curve below where the patients fails to benefit and upon which patients respond favorably to the treatment, so it is important to quantify the amount of MSC per ml that are injected [15]. MSC can be enriched through better aspiration and a harvesting technique and the number of MSC can then be maximized through centrifugation (Figure 55.4).

**Figure 55.4** Historical development of MSC in orthopedic surgery.

Ethical Issues

Stem cells have been a controversial topic since research on stem cells began with ESC lines. Controversy arose from the ethics of research involving the development, use and destruction of human embryos. Ethical concerns put a hold on further research. MSC, however, do not involve creating, using or destroying human embryos. MSC are autologous cells obtained from tissues of the same patient and are free from the ethical problems that affect the investigation and clinical use of embryogenic stem cells.

Harvesting and Processing of Mesenchymal Stem Cells

MSC can be aspirated from bone marrow in the pelvis at the iliac crest or can be obtained by lipoaspirate of abdominal fat or from the fat of the trunk with a liposuction procedure (Figure 55.5).

**Figure 55.5** Aspiration from iliac bone for harvesting RBM.

Red Bone Marrow (RBM) can be subdivided into two parts:

Hematologic, containing stem cells and already known since 1950
Stromal part, also containing precursors. The stromal fraction was defined early in 1869 by Goujon and shown in the 1970s by Friedenstein and Owen.

Havesting Bone Marrow Stem Cells

Bone marrow stem-cell aspiration can be performed either at the anterior or posterior iliac crest under fluoroscopic or ultrasound (US) guidance [16]. The area of the puncture is sterilized with a chlorhexidine solution. Ten millileters of 2% lidocaine are injected subcutaneously up to the iliac crest. A small incision is performed on the skin to allow the pass of an 11G trocar to the iliac crest. The trocar is advanced 2.5–5 cm and is inserted at the iliac crest and the stylet is withdrawn from the trocar. Bone marrow is aspirated through a syringe connected to the trocar. A total amount of 60 ml of bone marrow is aspirated. Hernigou et al. found that using rapid pulls with a 10 cc syringe yielded more MSC than slow pulls of 50 cc syringes [17]. Creation of negative pressure (suction) through a fenestrated needle ruptures vessels within the marrow and releases MSC. Blood from broken vessels is filtered through the tissue into the needle/syringe [18].

Concentration of Bone Marrow

After aspiration, bone marrow is immediately filtered and is submitted to a centrifugation process. The centrifugation forces the polynuclear cells characterized by the presence of heavier nuclei to be in the periphery where they can be removed.The lighter anuclear red cells are found in the center and can also be removed. Only the mononuclear cells (with stem cells) are spared and used for grafting.

Harvesting Adipose Tissue Stem Cells

Alternatively, for fat mesenchymal cell aspiration, a small incision is performed in the anterior abdominal wall or in the lumbar region (Figure 55.6).

**Figure 55.6** Patient preparation for fat liposuction at the flank abdominal region.

A 17G long needle with a cannula is inserted through the incision. A Klein solution of 25 ml of 2% lidocaine, 250 ml of normal and 1 ml epinephrine saline are injected in the subcutaneous fat through the 17G needle. Gentle agitation technique is applied to the abdominal wall or lumbar region while the emulsification takes place. Liposuction of the subcutaneous fat is performed using brisk-broad strokes while the luer-lock is pulled back to perform pressure to pull fat in the syringe; a total of 80 to 120 mg aspirate being obtained. The fat is then transferred in test tubes for processing and the liposuction procedure can be repeated to obtain higher amounts of fat (Figure 55.7).

**Figure 55.7** Sterile environment for tube processing.

**Figure 55.8** Microstructure of adipose tissue.

**Figure 55.9** Adipose harvest from liposuction.

**Figure 55.10** **(a)** and **(b)** show preparation of plastic tubes filled with adipose tissue obtained from liposuction and prepared for processing.

Adipose Stem Cell Enzymatic Digestive Process

Tissue samples obtained from liposuction are digested in a buffer solution containing collagenase with intermittent shaking. The digested solution is centrifuged and separated into the extracellular matrix with the oil in the upper layer and the cell layer precipitated at the bottom. The precipitated layer is called the stromal vascular fraction (SVF), which contains adipose stem cells [19, 20] (Figures 55.8–55.11).

**Figure 55.11** Reproduction of the fat process harvested by the liposuction procedure and undergone to cell enzymatic digestive process with collagenase (Roche®) for mesenchymal stem-cell injection.

Injection Techniques

Injection therapy is a very useful therapy and it is widely recommended for musculoskeletal disorders and it is used following international guidelines due to its relative safety, ease of application in trained hands and cost-effectiveness.

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