**4. Cartilage regeneration methods**

In 1959, abrasion arthroplasty was developed by Pridie to address chondral defects (Pridie, 1959). Originally developed as an open procedure, it was later adapted for arthroscopy by Johnson (Johnson, 1986). Today, abrasion arthroplasty is used primarily for osteoarthritic knees. However, for small focal chondral defects, microfracture is most commonly used.

Microfracture is a marrow stimulating technique that penetrates the subchondral bone in the cartilage defect (Fig 4). This allows marrow to communicate with the cartilage defect populating it with MSC, inflammatory mediators, and blood. This technique is simple to perform and has good to excellent clinical results (Gill, 2000; Steadman et al, 2003; Asik et al, 2008). Postoperative management requires prolonged non-weight-bearing (4 to 6 weeks) followed by the use of continuous passive motion. However, microfracture generates fibrocartilage in the defect and has a shorter functional lifespan compared with hyaline cartilage (Menche et al, 1996; McGuire et al, 2002; Steinwachs et al, 2008).

Fig. 4. Marrow stimulation with: (A) microfracture provides a source of (B) blood cells and bone marrow mesenchymal stem cells for cartilage regeneration. Healing response typically results in fibrocartilage formation.

Other techniques to address focal cartilage defects are osteoarticular transfer system with auto/allograft transplantation (OATS). This technique involves transplanting the defect with an intact cartilage and subchondral bone plug. This technique is typically used for small to medium sized lesions (0.5-3cm2) (Fig 5). If a larger area is to be addressed a mosaicplasty is performed with multiple plugs. The main disadvantages are donor site morbidity, breakdown between the implanted cartilage and subchondral bone, gaps that remain between plugs, as well as technical difficulty.

More recently autologous chondrocyte implantation (ACI) was developed to regenerate cartilage closer to hyaline cartilage. This technique can be used for larger defects (2-10 cm2), in patients who are symptomatic, and primarily located on the femoral condyles. To perform ACI requires 2 stages. The first stage involves harvesting cartilage from a biopsy to acquire cartilage cells. These cells are cultured to produce expanded autologous chondrocytes, which are subsequently implanted in the defect and held in place with a periosteal patch or collagen sheet sewn in place and sealed with a fibrin glue (Gooding et al, 2006). The postoperative course is challenging with a prolonged course of protected weightbearing and continuous passive motion for 4 to 6 weeks. It can take up to 1-1.5 years for larger lesions to fill in. Second generation ACI is currently undergoing development to overcome the technical disadvantages of the first generation. Second generation ACI uses

Articular Cartilage Regeneration with Stem Cells 135

term results and stability of the repair tissue remain to be demonstrated (Steinert et al, 2007). Stem cell transplantations are widely used in treatments where they are induced to differentiate into specific cell types required in repairing damaged or destroyed cell tissues (Chung et al., 2006). Stem cells are capable of differentiating in vitro and in vivo along multiple pathways that include bone, cartilage, cardiac and skeletal muscle, neural cells, tendon, adipose, and connective tissue (Panagiota et al., 2005). There has been a vast and increasing interest in the research and application of human mesenchymal stem cells (hMSC) in the area of regenerative medicine over the last decade. The reasons are due to the multipotency and stability of the cellular characteristics of hMSC. However, stem cell growth and differentiation, particularly for hMSC, requires a complex and tightly regulated

hMSC has been studied for many years and there are a set of tests that can be performed as the minimal criteria for defining multipotency of mesenchymal stromal cells (Dominici et al,

1. Plastic adherent assessment: hMSC is known to be able to adhere to plastic and hence the first observation is whether the cultured hMSCs exhibit the "plastic-adherent"

3. Multipotent differentiation potential assessment: One of the criteria that defines hMSC is that the cells must differentiate under in vitro culture conditions to osteoblasts, adipocytes and chondroblasts. The identification of these differentiated cells can be done via Alizarin Red or von Kossa staining (for osteoblast), Oil Red O (for adipocytes) and Alcian blue or immunohistochemical staining for collagen type II

There are numerous reports in the literature using MSC to regenerate bone and cartilage. A few of the examples are outlined in Table 1 below. Generally, the use of MSC will involve either direct injection or incorporation with a scaffold or matrix to aid delivery of the cells to the intended site (Nöth et al, 2008). Nevertheless, not all cases use MSC directly, most often the MSC are differentiated in vitro for various purposes. This involves some form of benchtop work either with isolation of cells or culture expansion thus leading to increased

The literature has not shown any strong evidence that any of the above methods actually generated fully function cartilage matching the original cartilage although evidence has suggested an improvement of the condition with these treatments. It is theorized that MSC have an unknown number of bioactive molecules that are immunoregulatory and able to promote regenerative activities (Chen et al 2006 & Uccelli et al, 2007). There are cases whereby MSC are used with or without a matrix as a vehicle to deliver gene therapy site specific. However, these are still experimental and will not be covered in this chapter. Additional approaches to regenerate hyaline cartilage are to adjunct bone marrow stimulation with growth factors and hyaluronic acid which is rich in glycosaminoglycans and provide the building blocks necessary for cartilage regeneration. Investigators are beginning to evaluate platelet rich plasma (PRP). There are numerous proteins found in platelets including growth factors involved in the healing response such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth

behaviour using standard tissue culture flasks at standard culture conditions. 2. Surface antigen expression: more than 95% of the hMSC population is to express positive for CD105, CD73 and CD90. These can be easily measured using flow cytometry. In addition, the cells have to show negative expression (less or equal to 2%)

of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA class II.

system consisting of medium, growth factors and serum components.

2006). These are:

(for chondroblasts).

costs.

tissue engineered 3- dimensional scaffolds that are seeded with autologous chondrocytes to promote cartilage regeneration. Despite continued technological improvements, clinical outcomes have yielded primarily symptomatic relief with fibrocartilage generation. The regeneration of long lasting hyaline cartilage continues to be a challenge (Ossendorf et al, 2007; Tuan, 2007).

Fig. 5. Transfer system with auto/allograft transplantation (OATS) is illustrated in this figure with (A) harvesting and (B) second-look at one year showing chondral defects between the osteochondral plugs.

#### **5. Adjuncts to current methods**

Recent literature has illustrated that postoperative additions of HA to arthroscopic cartilage procedures yield better results (Johnson 1986; Kujawa & Caplan 1986; Gill, 2000; Freedman et al, 2004; Jakobsen et al, 2005; Gooding et al, 2006; Lee et al, 2007; Kang et al, 2008; Horvai et al, 2011; Fortier et al, 2011). High molecular weight hyaluronic acid is a component found in synovial fluid that has been investigated as adjunct for full thickness cartilage injury, microfracture, mesenchymal stem cells, and osteochondral allografting. Studies indicated that hyaluronic acid may play a role with differentiation of stem cells to chondrocytes, decreased joint inflammation, increased proteoglycan content, improved histologic scores, defect filling and incorporation, as well as decreased friction in the joint.

Another approach in attempts to regenerate hyaline cartilage are to use cells delivered either seeded on a matrix or transplanted to the defect similar to ACI. There are many cells that have been investigated, these include: mesenchymal stem cells (adipose versus bone marrow derived), chondrocytes, periostium, perichondrium. Short term results for these cellular based treatment modalities have been positive. However, long term clinical results are uncertain and have had limitations. Chondrocytes for example, lose chondrocyte phenotype with monolayer cell culture expansion and change to a fibroblastic appearance.

When chondrocytes are combined with matrix/scaffold materials the result is predominately fibrocartilage. Perichondral/periosteal cells also have chondrogenic potential in vitro and in vivo when seeding matrices. However limitations have included, variable results, the need for 2 surgeries for harvest and implantation, and instability of repair tissue. Mesenchymal stem cells either adipose derived or bone marrow derived have multilineage potential and in the appropriate microenvironment will differentiate to chondrocytes. In vivo studies have yielded short term success in generating hyaline cartilage. However, long

tissue engineered 3- dimensional scaffolds that are seeded with autologous chondrocytes to promote cartilage regeneration. Despite continued technological improvements, clinical outcomes have yielded primarily symptomatic relief with fibrocartilage generation. The regeneration of long lasting hyaline cartilage continues to be a challenge (Ossendorf et al,

Fig. 5. Transfer system with auto/allograft transplantation (OATS) is illustrated in this figure with (A) harvesting and (B) second-look at one year showing chondral defects

Recent literature has illustrated that postoperative additions of HA to arthroscopic cartilage procedures yield better results (Johnson 1986; Kujawa & Caplan 1986; Gill, 2000; Freedman et al, 2004; Jakobsen et al, 2005; Gooding et al, 2006; Lee et al, 2007; Kang et al, 2008; Horvai et al, 2011; Fortier et al, 2011). High molecular weight hyaluronic acid is a component found in synovial fluid that has been investigated as adjunct for full thickness cartilage injury, microfracture, mesenchymal stem cells, and osteochondral allografting. Studies indicated that hyaluronic acid may play a role with differentiation of stem cells to chondrocytes, decreased joint inflammation, increased proteoglycan content, improved histologic scores,

Another approach in attempts to regenerate hyaline cartilage are to use cells delivered either seeded on a matrix or transplanted to the defect similar to ACI. There are many cells that have been investigated, these include: mesenchymal stem cells (adipose versus bone marrow derived), chondrocytes, periostium, perichondrium. Short term results for these cellular based treatment modalities have been positive. However, long term clinical results are uncertain and have had limitations. Chondrocytes for example, lose chondrocyte phenotype with monolayer cell culture expansion and change to a fibroblastic appearance. When chondrocytes are combined with matrix/scaffold materials the result is predominately fibrocartilage. Perichondral/periosteal cells also have chondrogenic potential in vitro and in vivo when seeding matrices. However limitations have included, variable results, the need for 2 surgeries for harvest and implantation, and instability of repair tissue. Mesenchymal stem cells either adipose derived or bone marrow derived have multilineage potential and in the appropriate microenvironment will differentiate to chondrocytes. In vivo studies have yielded short term success in generating hyaline cartilage. However, long

defect filling and incorporation, as well as decreased friction in the joint.

2007; Tuan, 2007).

between the osteochondral plugs.

**5. Adjuncts to current methods** 

term results and stability of the repair tissue remain to be demonstrated (Steinert et al, 2007). Stem cell transplantations are widely used in treatments where they are induced to differentiate into specific cell types required in repairing damaged or destroyed cell tissues (Chung et al., 2006). Stem cells are capable of differentiating in vitro and in vivo along multiple pathways that include bone, cartilage, cardiac and skeletal muscle, neural cells, tendon, adipose, and connective tissue (Panagiota et al., 2005). There has been a vast and increasing interest in the research and application of human mesenchymal stem cells (hMSC) in the area of regenerative medicine over the last decade. The reasons are due to the multipotency and stability of the cellular characteristics of hMSC. However, stem cell growth and differentiation, particularly for hMSC, requires a complex and tightly regulated system consisting of medium, growth factors and serum components.

hMSC has been studied for many years and there are a set of tests that can be performed as the minimal criteria for defining multipotency of mesenchymal stromal cells (Dominici et al, 2006). These are:


There are numerous reports in the literature using MSC to regenerate bone and cartilage. A few of the examples are outlined in Table 1 below. Generally, the use of MSC will involve either direct injection or incorporation with a scaffold or matrix to aid delivery of the cells to the intended site (Nöth et al, 2008). Nevertheless, not all cases use MSC directly, most often the MSC are differentiated in vitro for various purposes. This involves some form of benchtop work either with isolation of cells or culture expansion thus leading to increased costs.

The literature has not shown any strong evidence that any of the above methods actually generated fully function cartilage matching the original cartilage although evidence has suggested an improvement of the condition with these treatments. It is theorized that MSC have an unknown number of bioactive molecules that are immunoregulatory and able to promote regenerative activities (Chen et al 2006 & Uccelli et al, 2007). There are cases whereby MSC are used with or without a matrix as a vehicle to deliver gene therapy site specific. However, these are still experimental and will not be covered in this chapter.

Additional approaches to regenerate hyaline cartilage are to adjunct bone marrow stimulation with growth factors and hyaluronic acid which is rich in glycosaminoglycans and provide the building blocks necessary for cartilage regeneration. Investigators are beginning to evaluate platelet rich plasma (PRP). There are numerous proteins found in platelets including growth factors involved in the healing response such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth

Articular Cartilage Regeneration with Stem Cells 137

BMPC (Ceselli et al, 2009). In addition, when injected subcutaneously into mice, these cells were found to migrate to multiple organs and integrate and function as the surrounding cells (Ceselli et al, 2009). In addition to implementing evidence from recent animal studies, we have sought to make use of clinical evidence regarding the potential and safety of PBPC, preferring to use PBPC as opposed to BMPC because of the ease of harvest, decreased harvest-site morbidity, and increased potential with these cells (Ceselli et al, 2009; Holig et al, 2009 & Ordemann et al, 1998). In the clinical setting, we prefer to use PBPC as opposed to BMPC due to the ease of harvest and the increased potential with these cells. Subsequently, we have developed a method involving standard marrow stimulation in the form of subchondral drilling and novel postoperative intraarticular injections of autologous PBPC in combination

Arthroscopic surgeons are regularly faced with the challenges of providing a satisfactory end result for the treatment of chondral lesions. Prior to the development of the current method, the first author was treating chondral lesions with standard subchondral drilling followed by postoperative intraarticular injections of hyaluronic acid (HA). This being a variant of marrow stimulation technique, produces fibrocartilage which is not as resilient as the original hyaline cartilage. The newly regenerated fibrocartilage gradually deteriorated with time. Since Year 2005, the first author has been dissatisfied with the inconsistent end

Fig. 6. Intraoperative view after subchondral drilling of the lateral patella facet - right knee (A). Second-look at 18 months (B) showing partial coverage of the defect with fibrocartilage. At the same time, veterinary surgeons have shown that injections of bone marrow aspirate into race horse's flexor tendon injuries resulted in satisfactory healing (Pacini et al, 2007; Taylor et al, 2007; Thomas et al, 2008 & Violini et al, 2009). This gave rise to the idea of utilizing stem cells in the knee joint to initiate articular cartilage repair after subchondral

A literature search suggests that the mesenchymal stem cell (MSC) is a better alternative to the chondrocyte as it is a less differentiated cell and is capable of differentiating into both bone and articular cartilage. As most chondral lesions involve both these components, cells that are capable of forming both bone and cartilage should theoretically

results following this method of cartilage repair as shown in Fig 6.

with HA to regenerate articular cartilage.

**7. Authors preferred method** 

**7.1 Evolution of technique** 

drilling.

factor (TGF-M1), fibroblast growth factor (FGF). Early research has shown that mesenchymal stem cells and chondrocytes exposed to PRP have increased cell proliferation and production of proteoglycans and collagen type II (Fortier et al, 2011). In a clinical cohort comparing hyaluronic acid with PRP injections, PRP had improved pain scores (Sanchez et al, 2008). However, the quality and longevity of the repair tissue generated by the adjunct of PRP is still unproven.


Table 1. Examples of clinical applications of hMSC
