**2. Stem cells used for cartilage regeneration**

Stem cells have multidifferentiation potential, which can differentiate into distinctive end-stage cell types including bone, cartilage, muscle, bone marrow stroma, tendon/ligament, fat, dermis, and other connective tissues [9]. There are many cell types that have been manipulated *in vitro* and subsequently implanted to repopulate a cartilage defect. It must be ensured that the implanted cells are immunoprivileged or provide immunosuppressive agents to avoid rejection by the host immune system.

#### **2.1 Autologous chondrocytes**

 Autologous chondrocytes were first used for the treatment of cartilage defects of the patients by a Swedish group in 1994 [10]. This approach needs a slice of healthy articular cartilage obtained arthroscopically from proximal part of the medical femoral condyle of the affected knee joint during the first operation [11]. The chondrocytes were isolated from this healthy articular cartilage and cultured for 2–3 weeks to prepare enough cells (about 5 × 106 ) for damaged cartilage repair [11]. The clinical studies have shown that the treatment of autologous chondrocytes prompts pain reduction, improves quality of life, and delays the need of joint replacement in many cases [12–14]. Despite the encouraging clinical results, there are still limitations to the use of autologous chondrocyte transplantation. The conventional technique is accompanied with periosteum harvest and fixation over the cartilage defects via large skin incisions. Autologous chondrocytes were injected underneath the periosteal flap. Hypertrophy of the periosteum with high rate of revision arthroscopies and the risk of transplant failure of up to 20% are major drawbacks of the conventional autologous chondrocyte transplantation [14]. Moreover, the complexity and the cost of the two surgical procedures, the biological response of the periosteal flap, and the de-differentiation and consequent capacity loss associated with *in vitro* expansion of isolated chondrocytes are also the limitations [15].

#### **2.2 Bone marrow-derived mesenchymal stem cells (BMSCs)**

Mesenchymal stem cells (MSCs) are multipotent stromal cells first identified and described in 1966 by Alexander Fridenstein [16, 17]. Adult MSCs were originally isolated from bone marrow in 1999 by Pittenger and his colleagues [18]. Subsequent studies have demonstrated that MSCs present in various parts of the body including bone marrow (BM), peripheral blood, umbilical cord blood, fatty tissues, skeletal and cardiac muscles, Wharton's Jelly of umbilical cord, facet joints, interspinous ligaments, and ligamentum flavum [19–23]. Many studies have shown that MSCs can migrate to injury sites, induce peripheral tolerance, and inhibit the release of

#### *Current Tissue Engineering Approaches for Cartilage Regeneration DOI: http://dx.doi.org/10.5772/intechopen.84429*

proinflammatory cytokines. It has been demonstrated that MSCs can also promote tissue repair and survival of damaged cells [24]. However, it is not clear which adult tissue-derived MSCs should be used as a good source for cartilage repair.

 Autologous bone marrow mesenchymal stem cell (BMSCs) transplantation was first used for the repair of full-thickness articular cartilage defects in human patellae by a Japanese group [25]. BMSCs were aspirated from iliac crest and the nucleated cells were cultured. Adherent cells were subsequently collected, embedded in a collagen gel, transplanted into the articular cartilage defect in patellae, and covered with autologous periosteum. Six months after transplantation, clinical symptoms (pain and walking disability) were improved and the improvement was persisted for 9 years post-transplantation [26]. Sixteen years after transplantation, no clinical problem has been reported. Human autologous BMSCs have been used successfully to treat articular cartilage defects. Twelve months after BMSC transplantation, magnetic resonance imaging (MRI) revealed complete defect fill and complete surface congruity with native cartilage [27]. Currently, autologous BMSC transplantation has been widely used for cartilage repair [26, 28, 29]. Although BMSC treatment did not require any cell expansion or manipulation, reducing costs, and risks involved, the quantity of bone marrow cells was somewhat unsatisfactory [16].

### **2.3 Adipose-derived stem cells (ADSCs)**

Among MSCs, adipose-derived stem cells (ADSCs) have been recognized as an appropriate cell type with chondrogenic potential and high proliferation capacity [30, 31]. Approximately 400,000 liposuction surgeries are performed in the USA each year, and these procedures yield anywhere from 100 ml to 3 liters of lipoaspirate tissue [32]. This material is routinely discarded. It is well known that adipocytes are developed from mesenchymal cells via a complex cascade of transcriptional and non-transcriptional events that occur throughout the human life. Thus, adipose tissue is a good stem cell source.

 The initial methods to isolate cells from adipose tissue were developed by Rodbell and colleagues [33]. They isolated adipose-derived stromal cells from rat fat pads by four steps. Step 1: Adipose tissue was minced into small pieces. Step 2: The adipose tissue pieces were digested with collagenase. Step 3: The cell pellet was obtained by centrifuge. Step 4: The cell pellet was cultured for future use. This protocol has been widely used for the isolation of adipose-derived stem cells (ADSCs) from human adipose tissues with some modifications [34, 35].

The adipose tissue can be collected by needle biopsy or liposuction aspiration. The collected adipose tissues should be washed with 5% penicillin/streptomycin (P/S)-containing phosphate-buffered saline (PBS) twice, and then the tissue samples should be put in a sterile tissue culture plate and cut into small pieces. The minced tissues are digested with 0.075% collagenase at 37°C for 30 min; the collagenase is removed by centrifuging the digested solution (adipose tissue and collagenase mixture) at 1200 g for 10 min; the adipose-derived stem cells-containing pellet is then resuspended with culture medium (alpha-MEM, Mediatech, Herndon, VA) supplemented with 20% of fetal bovine serum (FBS), 1% L-glutamine (Mediatech, Herndon, VA), and 1% penicillin/streptomycin (Mediatech, Herndon, VA). The cell suspension is filtered through 70-μm cell strainer and cultured in a humidified tissue culture incubator at 37°C with 5% CO2. The medium is changed every second day until the cells reach 80–90% confluence. It is important that the adipose tissue should be treated within 24 hours, and the cells isolated from about 500 mg of adipose tissue should be added into one well of 12-well plates.

Adipose-derived stem cells (ADSCs) are readily accessible with no morbidity and display the capability to differentiate into several cell lineages, including the

spontaneous chondrogenic differentiation [30]. Compared with bone marrowderived MSCs, adipose-derived MSCs from lipoaspirates are acquired using a less invasive procedure and are in large amounts [36]. ADSCs have been used for the repair of articular cartilage defect in nonweight-bearing areas [37].

#### **2.4 Synovial-derived stem cells (SDSCs)**

Synovial-derived MSCs have been isolated from human synovial fluid and synovium of the knee and the hip using the following protocols [38, 39]. The synovial tissue samples (wet weight 10–50 mg) were obtained aseptically from the joints and rinsed twice with Hanks' balanced salt solution (HBSS; Life Technologies, Carlsbad, CA) supplemented with antibiotic-antimycotic solution (100 units/ ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B; Life Technologies; Carlsbad, CA). The washed tissues were minced into small pieces and digested with 0.5 ml of 0.2% collagenase (Life Technologies, Carlsbad, CA) in highglucose Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies, Carlsbad, CA) at 37°C for 1 hour. The digested solution were removed by centrifugation at 1500 g for 10 min; the SDSCs-containing pellet was resuspended in growth medium (high-glucose DMEM supplemented with 10% FBS and 100 units/ml penicillin, 100 μg/ml streptomycin) and cultured in a humidified tissue culture incubator at 37°C with 5% CO2. The medium was first changed at day 7 and changed every 3 days until the cells reach 80–90% confluence. It is important that the synovial tissue should be treated within 24 hours.

SDSCs obtained by above procedures have a higher proliferative capacity and chondrogenic potential than the MSCs derived from other sources [39, 40]. A small synovial tissue biopsy is an easily accessible source of autologous MSCs in the context of an explorative or therapeutic arthroscopy. These cells can be subsequently used for the regeneration of damaged cartilage. Autologous chondrocyte transplantation used for cartilage defect repair is limited by the availability of cells, particularly in elderly individuals, and by the well-known dedifferentiation events associated with chondrocyte expansion [39, 41]. Furthermore, SDSCs can be harvested relatively in a minimally invasive manner from synovial fluid and retain a particularly high capacity for chondrogenic differentiation and proliferation compared with MSCs obtained from other tissues, such as bone marrow or cartilage, those have second injury on healthy tissues. SDSCs may be an optimal alternative source of chondrogenic cells for cartilage defect repair.

A recent research has shown that xenogenic implantation of equine SDSCs into rat cartilage defect area leads to articular cartilage regeneration [42]. Horse joints are anatomically equivalent to the human knee and ankle; as a result, horses are widely used as large animal preclinical models for cartilage repair studies. However, large animal studies pose logistical and financial challenges, and small animal rodent models are cost-effective and have proven to be useful for proof-of-concept studies. There was no any immune response to the equine cells in the treated rat knees [42]. This result was also confirmed by a xenogenic transplantation of human MSCs in a critical size defect of the sheep tibia for bone regeneration [43]. Another xenogenic transplantation study has shown that human MSCs can enhance damaged pig intervertebral disc regeneration [44].
