**9. Conclusions**

A cartilage repair treatment using tissue engineering comprises the implantation of bioabsorbable scaffolds that at first fill a chondral or osteochondral defect, then the production of cartilage repair tissue depends on the *de novo* synthesis of cartilage matrix elements. Such scaffolds support the local migration of cells (chondrogenic or osteogenic) that basically synthesize new extracellular matrix. The aim of all cartilage replacement strategies should focus on reconstruction of hyaline cartilage with its hierarchical organization; however, most of the current strategies based on monophasic designs lead to the production of fibrocartilage, which has inferior biological and mechanical characteristics compared to hyaline cartilage.

 The design of multiphasic scaffolds aims at congruence with that of hierarchical nature, and from the studies that have been carried out over the past few years, it is clear that as a consequence, it substantially improves the integration of the implant with the surrounding osteochondral tissue, and positively influences the functional regeneration of both chondral and bone tissues. A vast array of multiphasic designs has been evaluated in vitro; however, only three are currently available in the clinic; the question that arises is: how to optimize the efforts to achieve a conclusive clinical application?

The use of scaffolding in order to recapitulate as much as possible the hierarchical structure seems to be not enough. The decision to cellularize or maintain a cell-free scaffold is crucial, and the answer will depend on the 3D system in a particular way; therefore, cellularization in each of the chondral and bone phases must be taken into account for the final design. On the other hand, the inclusion of an in vitro maturing time of the cellularized implant is desirable; thus, at the time of implantation, the graft has enough mechanical characteristics to support the mechanical request in the joint.

 The needed to mimic the ECM on a molecular level is another main goal that demands to be taken into account, so the bioactivation of the biomaterials with elements such as synthetic materials as the ceramics (tricalcium phosphate, hydroxyapatite, and bioactive glass), or even the same decellularized tissue matrix, turns out to be a valuable tool for cartilage design, since these materials enhance the growth of a bone-like layer to support the overlying cartilage to the existing osteochondral defect.

Experimental studies are ongoing to evaluate innovative multiphase designs regarding the interaction with cells and the environment in an *in vivo* framework. *In vivo* trials using small animal models provide innovative concepts in osteochondral tissue engineering; nonetheless, to reach the development of clinical trials in humans, it is important to follow successful experiments using animal models that have loads and joint dimensions similar to humans. Animals such as sheep, pigs, and horses have surgically created defect sizes ranging from 0.29 to 0.79 cm<sup>2</sup> and have average human-like defects depths of about 0.68–1 cm. The body weights of these animals are also comparable or much heavier than humans, which makes them more appropriate models to predict the results in clinical trials.

Although the challenge to incorporate the use of multiphase designs to the clinic is still great, from the results observed in the wide range of studies, it is possible to conclude that tissue engineering approaches based on multiphasic scaffolds represent a promising therapeutic treatment for the regeneration of osteochondral defects. Moreover, based on the clinical results, it seems that a three-phase approach offers the most promising results with patients.
