**2. Therapeutic interventions without active biologics**

#### **2.1. Bone marrow stimulation**

A first group of interventions can be described as "marrow stimulating techniques". Drilling the defect beyond the subchondral plate is its essential and allowing a repair promoted by the bleeding from the subchondral bone creating a "super clot" its rationale (Mithoefer et al., 2005). The star cells here are the mesenchymal stem cells (MSCs) emerging with the hematoma and its subsequent proliferation and differentiation. Unfortunately, the final result is the promotion of a fibrous tissue not durable in time (Steret et al., 2004). Pridie et al. promoted this concept in 1959. Abrasion arthroplasty (Steret et al., 2004), microfracture (Steadman et al., 2003), or Autologous Matrix Induced Chondrogenesis (Gille et al., 2010) have been more

In essence, previous treatments have attempted to promote the healing of the damaged reminding nature. That is to say, allowing the flood of blood from the inner areas of the subjacent bone. Unfortunately, this will promote a scar tissue that in the long run will be lost

In a second group of interventions, it is proposed the articular defect to be covered by a "replacement technique". In these, the defect is reshaped to a standard cylindrical way and substituted with a plug of osteochondral graft harvested from a non-weight bearing donor site. Using this technique two handicaps can be appreciated: the limited available graft and the morbidity of the donor site. Additionally, the differences in characteristics between the donor and the receiver areas may impede a complete integration. This may be the reason why the clinical results have not been in accordance with the initial enthusiasm (Mishima et al., 2008). Both, auto- and fresh allograft (Gross et al., 2002) have been attempted with the name of mosaicplasty. With the aim of avoiding the donor site morbidity, synthetic reabsorbable scaffolds have been used to fill up the osteochondral defect. A "toast and butter" cylinder, engineered mimicking components, bone and cartilage. In contrast with the graft, this scaf‐ fold will have osteoconductive properties instead and eventually resorb in 12 months. The pseudo-cartilage now created will bepoorly incorporatedandthe biomechanics willfail(Yasen

The third group of proposed interventions are cellular-based. The aim in this alternative option is producing a regeneration of the cartilage mediated by the own patient chondrocytes. This is a two-stage procedure. During the preliminary intervention a biopsy from the cartilage is obtained and then the chondrocytes are isolated and cultured till the number of cells is about fifty folds. For the second intervention, *ex vivo* expanded chondrocytes are implanted in the damaged area. This procedure has evolved with the aim of getting a watertight seal environ‐ ment in order to receive the chondrocytes and avoid the leakage. First, it was attempted a sheet of periosteum, then a collagen gel and a collagen membrane has been developed. But, are the delivered chondrocytes, the MSCs coming from the subchondral bone or the cells evading from the layer of periosteum the source for promoting the repaired tissue?. In spite of the rationale of this techniques, the autologous chondrocytes implant has not finally reached the expected results, the final tissue obtained was fibrous instead of AC (Tins et al., 2005) and hence, the OA is once more the undesirable expected long-term result (Hunziker, 2002;

and the joint degenerative process will be stated (Kreuz et al., 2006).

recently developed with the same rationale.

158 Regenerative Medicine and Tissue Engineering

et al., 2012).

Temenoff et al., 2000).

First approaches to heal cartilage by *in situ* regeneration date back to 1959. Pridie technique was directed to BM cells recruitment to be used in cartilage defects by drilling small holes into the subchondral BM space underlying the damaged cartilage regions. It was improved later on by reducing the size of the perforations and being then called microfracture technique which is now a frequently performed and well-studied procedure (Steadman et al., 1999). This technique is based on the mechanism of mesengenesis or capacity of the non-differentiated mesenchymal cells in choosing a determined phenotype as a response to inducing or GFs. A non-differentiated cell from the BM can be promoted to different cell types such as osteoblasts, with a later maturing to osteocytes, chondroblasts and chondrocytes, but also to endothelial cells, mesothelial cells, fibroblasts or adipocytes. It is a cell signalling process of local cytokines on local cells. In order to achieve all this, the surgical technique is based on drilling the subchondral plate to get bleeding and a superclot that will become a scaffold and supply cells and proteins, starting this way the physiological cascade of the chondrogenic cell differentia‐ tion. Other alternative techniques of BM stimulation to regenerate cartilage would be abrasion chondroplasty and in case the articular surface remained untouched, the retrograde stimula‐ tion technique. Cartilage defects are repaired only with fibrous tissue or fibrocartilage when using these methods, probably because the number of chondroprogenitors recruited from the BM is too small to promote the hyaline cartilage repair and results are often followed by degeneration of the repair tissue. This was used as an explanation for the observations of other studies that good short term results may be followed by deterioration starting about 18 months after surgery.

Clinical observations and theoretical considerations pointed towards several possible limita‐ tions of marrow stimulation techniques. The non-adhesive properties of the cartilage surface and the softness and shrinking of the superclot can lead to only partial defect filling and facilitate an early loss of repair tissue from the cartilage lesion. To avoid this, the treatment has been recently advanced into a matrix-supported technique in which the performed defect was stabilized in an additional way with a biomaterial. The microfractured lesion is covered with a collagen type I ⁄ III scaffold and it is called autologous matrix induced chondrogenesis (AMIC) (Kramer et al., 2006; Steinwachs et al., 2008). This technique has been developed to allow the treatment of larger defects by microfracturing and it is used as alternative treatment to autologous chondrocytes transplantation (ACT).

#### **2.2. Autologous osteochondral transplantation: Mosaicplasty**

Autologous osteochondral mosaicplasty, sometimes known as osteoarticular transfer system, OATS, is an effective method for the resurfacing of osteochondral defects of the knee. The technique consists in transplantation of many osteocondral autologous plugs obtained from the periphery of the femoral condyle articular surface, which supports less weight and transferring them to create a durable resurfaced area in the defect (Fig. 3). The procedure shows some advantages regarding other repair techniques, such as the viable hyaline cartilage transplantation, a relatively short rehabilitation period and the possibility of carrying out the procedure in one only operation.

**Figure 3.** In mosaicplasty cylindrical osteochondral plugs are harvested from nonload-bearing sites in the affected joint and pressed into place within the osteochondral defect, creating an autograft "mosaic" to fill the lesion

However, the OATS limitations are the donor-site morbidity and a limited availability of grafts that can be obtained from the femoropatellar joint or the area adjacent to the intercondylar fossa. Other possible limitations are differences in bearing, thickness and mechanical proper‐ ties between the donor's and the receiver's cartilages, as well as the graft sinking into the surface due to the support of weight after surgery. Besides, the lack of filling and the possible dead space between cylindrical grafts can limit the repair quality and integrity. Lane et al. transplanted autologous osteochondral grafts into sheep knee joints and reported the lack of integration of the cartilage, which determined the persistence of gaps through the full thickness in all the specimens (Mishima et al., 2008).

#### **2.3. Alogenic osteochondral transplantation**

Osteochondral allograft transplantation is a procedure for cartilage resurfacing which involves the transplantation into the defect a cadaveric graft composed of viable, intact AC and its underlying subchondral bone. It is a well known resource, especially for tumour surgery. The defect size, its location and its depth are crucial factors for the suitability of the donor graft. Advantages of using osteochondral allografts are the possibility if achieving a precise archi‐ tecture of the surface, the immediate transplantation of viable hyaline cartilage in a one-time procedure, the possibility to repair large defects, even half-condyles and the donor-site lack of morbidity. Gross et al. have reported results from fresh allografts in 123 patients with good clinical results in 95% of the patients after five years (Gross et al., 2002). There are different possible allografts. Fresh osteochondral allografts are generally used because both freezing and cryopreservation have proved to reduce the chondrocytes viability. Traditionally grafts have been obtained, kept in lactated Ringer's solution at 4 °C and then transplanted in a week. Another alternative for allografts conservation and implantation is cryopreservation, which involves freezing at a controlled speed of specimens within a nutrients rich medium, a cryoprotector agent (glycerol or dymethil sulfoxide), to minimize the cells freezing and keep their viability; finally, there is the possibility of fresh-frozen allografts, with the advantages of lower immunogenic capacity and less transmission of diseases but with lower chondrocyte viability.

#### **2.4. Soft tissues transplantation**

transplantation, a relatively short rehabilitation period and the possibility of carrying out the

**Figure 3.** In mosaicplasty cylindrical osteochondral plugs are harvested from nonload-bearing sites in the affected joint and pressed into place within the osteochondral defect, creating an autograft "mosaic" to fill the lesion

However, the OATS limitations are the donor-site morbidity and a limited availability of grafts that can be obtained from the femoropatellar joint or the area adjacent to the intercondylar fossa. Other possible limitations are differences in bearing, thickness and mechanical proper‐ ties between the donor's and the receiver's cartilages, as well as the graft sinking into the surface due to the support of weight after surgery. Besides, the lack of filling and the possible dead space between cylindrical grafts can limit the repair quality and integrity. Lane et al. transplanted autologous osteochondral grafts into sheep knee joints and reported the lack of integration of the cartilage, which determined the persistence of gaps through the full thickness

Osteochondral allograft transplantation is a procedure for cartilage resurfacing which involves the transplantation into the defect a cadaveric graft composed of viable, intact AC and its underlying subchondral bone. It is a well known resource, especially for tumour surgery. The defect size, its location and its depth are crucial factors for the suitability of the donor graft. Advantages of using osteochondral allografts are the possibility if achieving a precise archi‐ tecture of the surface, the immediate transplantation of viable hyaline cartilage in a one-time procedure, the possibility to repair large defects, even half-condyles and the donor-site lack of morbidity. Gross et al. have reported results from fresh allografts in 123 patients with good clinical results in 95% of the patients after five years (Gross et al., 2002). There are different possible allografts. Fresh osteochondral allografts are generally used because both freezing and cryopreservation have proved to reduce the chondrocytes viability. Traditionally grafts have been obtained, kept in lactated Ringer's solution at 4 °C and then transplanted in a week. Another alternative for allografts conservation and implantation is cryopreservation, which involves freezing at a controlled speed of specimens within a nutrients rich medium, a cryoprotector agent (glycerol or dymethil sulfoxide), to minimize the cells freezing and keep

procedure in one only operation.

160 Regenerative Medicine and Tissue Engineering

in all the specimens (Mishima et al., 2008).

**2.3. Alogenic osteochondral transplantation**

Two main theories support the practicing of covering the cartilage defects with soft tissues, such as perichondrium or periosteum. On one hand, the defect has to be covered mechanically and on the other, we know about the presence of pluripotential stem cells in the perichondrium and the periosteum cambium layer. The different factors able to promote these cells differen‐ tiation into active chondrocytes still remain unknown.
