**3. Therapeutic interventions with active biologics**

#### **3.1. Autologous chondrocytes implant**

The clinical use of the autologous chondrocytes implant (ACI) technique was first reported by Brittberg et al. in 1994, following animal studies which had shown its effectiveness (Grande et al., 1989). In this method, chondrocytes are obtained from a biopsy taken from a non-weight bearing part of the patients cartilage, and are expanded *in vitro*, followed by the injection of a suspension of chondrocytes into cartilage defects, covered with autologous periosteal flap (Fig. 4). This technique premises are based on the capacity of adhesiveness of the cells to certain surfaces, they spread on them and proliferate producing their specific ECM. Although clinical results of the original ACI looked promising (Minas, 2001; Peterson et al., 2000), this procedure has some potential disadvantages, such as leakage of transplanted cells, invasive surgical method, hypertrophy of periosteum (Haddo et al., 2005; Kreuz et al., 2009) and loss of chondrogenic phenotype of expanded chondrocytes in monolayer culture (Benya & Shaffer, 1982). Second generation ACI, named membrane autologous chondrocyte implantation (MACI), has a similar procedure, but a collagen type I and III membrane instead of periosteum. This technique was introduced to improve the ACI problems, and biomaterials such as collagen type I gel (Ochi et al., 2002), hyaluronan-based scaffold (Manfredini et al., 2007) and collagen type I/III membrane (Bartlett et al., 2005) were applied to secure cells in the defect area, to restore chondrogenic phenotype by way of three dimensional cultures (Gigante et al., 2007]) and to replace the periosteum as defect coverage. This is the way MACI technique is created *a posteriori*, by implanting autologous chondrocytes in three dimensional matrices of collagen types I and III, or hyaluronic acid.

At present, only two prospective studies comparing the original and second generation ACI are available (Bartlett et al., 2005; Manfredini et al., 2007) and both studies show no significant differences in the short term clinical results. As for the first generation ACI, the newly regenerated cartilage often consists of fibrous tissue (Horas et al., 2003; Tins et al., 2005), possibly due to the limited number of chondrocytes and their low proliferation potential. Bone overgrowth that causes thinning of the regenerated cartilage and the violation of the tidemark are also of concern. Moreover, this method still sacrifices healthy cartilage. Thus, these aspects

**Figure 4.** In autologous chondrocytes implantation (ACI) a chondral biopsy is taken from a donor site at the time of clinical examination to be treated with enzymes in the laboratory to obtain chondrocytes cultures that are re-injected under the periosteal flap

limit ACI in the treatment of large defects and may increase the long-term risk of osteoarthritis development.

#### **3.2. Are Mesenchymal Stem Cells what we need?**

Stem cells are of particular interest in Regenerative Medicine. They inhere several unique characteristics that distinguish them from other cell types. Besides autograft transplantation and ACI, current therapeutic concepts of cartilage defects include the recruitment of MSCs. Tissue engineering (TE) based on cell and genetic therapy offers some of the most promising strategies of tissue repair, including AC repair. It is the science able to create alive tissue to replace, repair or strengthen ill tissue. Thus, the TE refers to a wide variety of techniques.

The process for using MSCs to produce cartilage tissue comprises four elements: cells, inductor factors, scaffold to deliver the cells and vascular supply to the host area.

From the previous experiences we have learned that we need:


differentiation capacity and it must be related to the special cytokines, growth factor and induction molecules composition of the medium (Claros et al., 2012; Hennig et al., 2007). To induce the differentiation of predifferentiated cells, MSCs, is a more controlled way of driving the process to a cartilage final effector. One of the cons of chondrocyte transplan‐ tation is the dedifferentiation process that these cells suffer when they are treated *in vitro* and the limited ability to redifferentiate them (Benz et al., 2002). On the contrary, MSCs are very stable and they do not suffer this dedifferentiation (Najadnik et al., 2010) process and have a high differentiation capacity.

Up to now, MSCs have been used to treat osteochondral defects in two different ways. We have the option of implanting the MSCs once expanded. It is of concern in this case the lack of control on the growth after transplantation and how much exhausted the cells will result after the expansion and how it will influence on the aging of the new induced tissue. The clinical results after implantation of these expanded MSCs have been analysed and were substantially the same compared with those obtained after any marrow stimulating techniques (Wakitani et al., 2002; López-Puerta, 2013). A second option is to implant the MSCs after the induced differentiation to a chondrocyte phenotype. In these circumstances, to assure the stability of the achieved phenotype is of concern as this lineage is able to go on with the complete chondral process and ends in producing final stages of hypertrophy and calcification (Pelttari et al., 2006; Andrades et al., 2010; López-Puerta, 2013). In any case, the final challenge will be to produce a stable cellular lineage that produces an AC tissue that works under compression forces without losing its initial characteristics as time goes by.

limit ACI in the treatment of large defects and may increase the long-term risk of osteoarthritis

**Figure 4.** In autologous chondrocytes implantation (ACI) a chondral biopsy is taken from a donor site at the time of clinical examination to be treated with enzymes in the laboratory to obtain chondrocytes cultures that are re-injected

Stem cells are of particular interest in Regenerative Medicine. They inhere several unique characteristics that distinguish them from other cell types. Besides autograft transplantation and ACI, current therapeutic concepts of cartilage defects include the recruitment of MSCs. Tissue engineering (TE) based on cell and genetic therapy offers some of the most promising strategies of tissue repair, including AC repair. It is the science able to create alive tissue to replace, repair or strengthen ill tissue. Thus, the TE refers to a wide variety of techniques.

The process for using MSCs to produce cartilage tissue comprises four elements: cells, inductor

**•** To minimize the risk and inconveniences of the donor site. In this case, MSCs are easily available from bone marrow, synovial membrane, adipose tissue, etc. So then, we can get a variable number of cells from a different tissue, partially avoiding the donor site secondary complication (Winter et al., 2003). In ACI, this extra procedure adds a risk for infection, inflammatory changes in the joint, pain and rises up the final cost of the treatment (Hunziker,

**•** To achieve a minimum number of cells. MSCs have a high proliferation and differentiation potential. In any case, MSCs coming from different tissue have an uneven chondrogenic

factors, scaffold to deliver the cells and vascular supply to the host area.

From the previous experiences we have learned that we need:

development.

under the periosteal flap

162 Regenerative Medicine and Tissue Engineering

2002)

**3.2. Are Mesenchymal Stem Cells what we need?**

Beside the characteristics of MSCs exposed before, these cells have self-renewal potential as well as multilineage differentiation potential (Becerra et al., 2011), including chondrogenesis (Johnstone et al., 1998; Pittenger et al., 1999; Prockop 1997; Sacchetti et al., 2007). MSCs chondrogenesis was first reported by Ashton et al. (1980) and the first ones to describe a defined medium for *in vitro* chondrogenesis of MSCs were Johnstone et al. (1998), who used micromass culture with TGF-β and dexamethasone. Sekiya et al*.* (2001, 2005) reported that addition to bone BMPs enhanced chondrogenesis under the conditions employed by Johnstone et al. (1998). Nowadays, the micromass culture is widely used to evaluate chondrogenic potential of MSCs *in vitro*. However, this *in vitro* chondrogenesis does not mimic cartilage formation during development. During micromass culture, MSCs increase expressions of both collagen type II (chondrocytes marker) and X (hypertrophic chondrocytes marker) (Barry et al., 2001; Ichinose et al., 2005). Other cytokines such as IGF (Pei et al., 2008) and parathyroid hormonerelated peptide (PTHrP) had been tried for better differentiation cocktails, but it is still difficult to obtain *in vitro* MSC-based cartilage formation comparative to native cartilage tissue.

To initiate any regeneration based on MSCs activity, the cells first have to be recruited to the site of damage (Fig. 5). Second step is adhesion to a local matrix followed by activation and extensive proliferation to provide the necessary high numbers of chondroprogenitor cells to build up new tissue. In stem 3, the cells need to switch from expansion to chondrogenic matrix production by induction of chondrogenesis to build up the shock absorbance and gliding characteristics for proper tissue function. The seamless integration with neighbouring cartilage and bone tissue depends on successful crosstalk between new and old tissue, and an instruc‐ tional capability to guide neighbouring cells. For durable cartilage regeneration, the tissue eventually needs to regenerate a tidemark, adapt to biomechanical loading and build up a balanced tissue homeostasis. We are going to summarize these steps including talking points taken from our annual meetings in NACRE (New Approaches for Cartilage Regeneration; CIBER-BBN, Spain) consortium, in order to propose strategies for the Regenerative Medicine of AC (Becerra et al., 2010; Andrades et al., in press)

**Figure 5.** Principal mechanisms for *in vivo* cartilage regeneration. 1) MSCs recruitment from the surrounding tissues such as synovial fluid/membrane, neighbors cartilage, or subchondral bone; 2) MSCs retention by local adhesion, and proliferation; 3) MSCs are induced to chondrogenic differentiation by local factors; 4) chondroblasts and chondrocytes arranged according to the cartilage zones pattern and biomechanical loading; 5) integration of repair tissue interac‐ tion with cartilage and subchondral bone

#### *3.2.1. Recruitment of MSCs*

Cell migration is a prerequisite for development from conception to adulthood and plays a major role in regeneration of all tissues. Even articular chondrocytes, which are encased in a dense matrix throughout their life, can show cell motility when transferred *in vitro.* A number of studies demonstrated that chondrocytes migrate under the action of different stimuli on or within planar and 3D matrices. Attracting factors include bone morphogentic proteins (BMPs) (Frenkel et al., 1996), insulin-like growth factor-one (IGF-1), transforming growth factor-beta (TGF-β), fibronectin (Andrades et al., 2003: Chang et al., 2003), plateled-derived growth factor (PDGF) (Fujita et al., 2004), fibroblast growth factor (FGF) (Hidaka et al., 2006), fibrin and collagen type I (Kirilak et al., 2006).

But, what are the best factors to attract more MSCs to a cartilage lesion? Recent work has established that MSCs are not largely distinct from chondrocytes regarding the panel of factors capable to attract the cells *in vitro.* All tested factors were further effective in stimulating the migration of MSCs but not fibroblasts (Ozaki et al, 2007). Factors contained in a natural blood clot are highly chemoattractive for MSCs; augmentation of a clot-stabilizing matrix with a potent chemoattractive factor like PDGF may be an attractive way to further enhance cell numbers early after microfracturing.

In this sense, a quick release kinetics within hours and days from the biomaterial is desired for this first step of healing. Unfortunately, no 3D *in vitro* model in a biomatrix has yet been applied to test chemoattraction of human MSCs under more natural conditions and no adequate animal model has been used to dissect the factor requirement of progenitor cell recruitment from bone marrow into cartilage defects. Most likely, the presence of synovial fluid and the joint loading initiated pumping mechanism will strongly affect attraction and retention of MSCs. A search for superior factor combinations to enhance and speed up MSC attraction, and the best retention matrix to keep cells local, thus, is an important topic for upcoming studies.

#### *3.2.2. Multiplication of MSCs*

tional capability to guide neighbouring cells. For durable cartilage regeneration, the tissue eventually needs to regenerate a tidemark, adapt to biomechanical loading and build up a balanced tissue homeostasis. We are going to summarize these steps including talking points taken from our annual meetings in NACRE (New Approaches for Cartilage Regeneration; CIBER-BBN, Spain) consortium, in order to propose strategies for the Regenerative Medicine

**Figure 5.** Principal mechanisms for *in vivo* cartilage regeneration. 1) MSCs recruitment from the surrounding tissues such as synovial fluid/membrane, neighbors cartilage, or subchondral bone; 2) MSCs retention by local adhesion, and proliferation; 3) MSCs are induced to chondrogenic differentiation by local factors; 4) chondroblasts and chondrocytes arranged according to the cartilage zones pattern and biomechanical loading; 5) integration of repair tissue interac‐

Cell migration is a prerequisite for development from conception to adulthood and plays a major role in regeneration of all tissues. Even articular chondrocytes, which are encased in a dense matrix throughout their life, can show cell motility when transferred *in vitro.* A number of studies demonstrated that chondrocytes migrate under the action of different stimuli on or within planar and 3D matrices. Attracting factors include bone morphogentic proteins (BMPs) (Frenkel et al., 1996), insulin-like growth factor-one (IGF-1), transforming growth factor-beta (TGF-β), fibronectin (Andrades et al., 2003: Chang et al., 2003), plateled-derived growth factor (PDGF) (Fujita et al., 2004), fibroblast growth factor (FGF) (Hidaka et al., 2006), fibrin and

But, what are the best factors to attract more MSCs to a cartilage lesion? Recent work has established that MSCs are not largely distinct from chondrocytes regarding the panel of factors capable to attract the cells *in vitro.* All tested factors were further effective in stimulating the migration of MSCs but not fibroblasts (Ozaki et al, 2007). Factors contained in a natural blood clot are highly chemoattractive for MSCs; augmentation of a clot-stabilizing matrix with a

of AC (Becerra et al., 2010; Andrades et al., in press)

164 Regenerative Medicine and Tissue Engineering

tion with cartilage and subchondral bone

collagen type I (Kirilak et al., 2006).

*3.2.1. Recruitment of MSCs*

Proliferation of MSCs is the second important step to rapidly enhance the cell numbers in the repair tissue. As the replicative lifespan of MSCs is, however, not unlimited and telomerase activity is absent or low (Parsch et al., 2004) this can only ground on the sufficient attraction of initial MSCs numbers to the defect. During embryonal development proliferative chondro‐ progenitors are densely packed and condensed to an area in which cartilage tissue is forming. The high cell numbers may be needed to deposit the vast amount of ECM characterizing this tissue. Low cellularity of AC is rather a late phenomenon during tissue formation and may have developed in adaptation to its biomechanical competence and the extremely slow turnover of its ECM. Proteoglycan turnover in cartilage is up to 25 years and collagen half-life was estimated to range from several decades up to 400 years (Eyre et al., 2006). Based on the assumption that embryonal traits should best be recapitulated during tissue regeneration, early defect filling tissue should contain densely packed proliferating chondroprogenitors. This would mean that rather hundreds of millions of new cells are needed per cm3 during this step in the defect to allow for optimal chondrogenesis and rapid ECM production. At a later phase, cell numbers could decline to a density of 5-10 million cells per gram tissue, when homeostasis is the left over task for the cells in the fully regenerated tissue.

These points towards an outstanding need for highly efficient transient induction of prolifer‐ ation, a typical feature ascribed to adult stem cells. Growth requirements of human MSCs are distinct from those of other species (Kuznetsov et al., 1996) and many factors have been identified as potent mitogens, being PDGF-BB, EGF and TGF-β have been regarded as the most important amongst them (Kuznetsov et al., 1997). As they induced the migration of MSCs and have at the same time the potency to enhance their proliferation, the migration and prolifera‐ tion steps of MSCs can take place simultaneously *in vivo.* However, whilst nutrients and oxygen are almost unlimited in tissue culture, a rapid supply of cells with O2 and nutrients may be more restricted in a cartilage defect and depend on the distance to and conditions found within the subchondral bone. Histology of early human cartilage repair tissue demonstrates that indeed cartilage differentiation initiates in contact with subchondral bone (Steck et al., 2009) whilst upper regions remain fibrous for quite a long time and may need to mature over years (Brun et al., 2008). Furthermore, earliest chondrogenesis is often seen in areas where active remodeling of the subchondral bone plate occurs and, thus, enhanced nutrition and a higher anabolic rate of the cells can take place. Beside strong mitogenic factors used for augmentation of a clot-stabilizing biomaterial, the access to optimal nutrition in the course of tissue remodeling may indeed be a limiting aspect of cartilage regeneration techniques. Enhanced remodeling of microfractured compared with unopened subchondral bone areas is likely, but quantitative and localization-dependent studies have so far not been reported. The stimuli mentioned here could be obtained by direct administration of recombining growth factors in the culture media or via transfer of the respective genes. Thus, the possibility of considering genetic therapy as an applicable measure for the treatment of cartilaginous lesions arises.
