**4. The recapitulation of AC morphogenesis through MSCs differentiation**

In spite of therapeutic strategies mentioned, which offer the patient a temporary relief of symptoms, they do not resolved, in the medium or long term, the disease that affects the joint. In most cases, fibrocartilage is generated that does not provide the necessary structural integrity and this often results in the subsequent replacement of the damaged joint (Mahmou‐ difar and Doran, 2012).

The main problem with these strategies is that only tries to restore the biomechanical charac‐ teristics of tissue but not its physiology. That is why the goal of current research in AC regeneration with MSCs is moving to methods that attempt to recapitulate the morphogenesis of the tissue. The challenge is to develop strategies that will cover the widest possible range of intervening factors including: the dynamics of genes and proteins that control and partici‐ pate in the chondrogenic process, their spatiotemporal patterns of expression, variations in culture conditions, biomaterials functionalized with effector molecules inducers of chondro‐ genesis and the inclusion of differentiation enhancers using genetic engineering and others.

#### **4.1. Novel genes and protein: The path to recapitulating AC morphogenesis**

As previously mentioned, the AC's ECM is composed mainly of collagen type II fibers and proteoglycans with strong negative charges, aggrecan being the most abundant of them (Han et al, 2011). That is why the detection of both proteins and their corresponding genes has been classicallyusedasmarkersof chondrogenesis innumerous studies *invitro* and*invivo*.However, most modern regenerative strategies should aim at obtaining a more complete and organized ECM that achieves the greatest similarity with the ECM on the original AC. With this objec‐ tive, have been identified dozens of genes that play a fundamental role in the formation and maintenance of AC, which are potential targets for research in the design of regenerative strategies (Quintana et al., 2009; Bobick et al., 2009; Mahmoudifar and Doran, 2012).

#### *4.1.1. Proteoglycan 4 (PRG4): Biological function and its relationship to OA*

A good example is the proteoglycan 4 (PRG4), also known as "superficial zone protein" (SZP) and "lubricin". It is a proteoglycan specifically synthesized by chondrocytes located at the surface of AC and by synoviocytes. Their functions are the lubrication of articular joints, elastic absorption and energy dissipation of synovial fluid (Jay et al., 2007).

It has been shown that mutant mice *Prg4*-/ - have normal joints at birth and during the newborn period but older mice exhibit accumulation of proteinaceous deposits on the cartilage surface, disappearance of the surface zone of flattened chondrocytes, synoviocyte hyperplasia, calcification of structures flanking the ankle joints and, consequently, a total failure of the joint (Coles et al., 2010). These data are consistent with OA degeneration, and have also been observed in veterinary cases of sheep with early OA which has been shown a downregulation of the expression of *PRG4* (Young et al., 2006). In a human clinical example, a case study reported in 2004 a 10 year old boy with Camptodactyly-Arthropathy-Coxa vara-Pericarditis syndrome (CACP) which arises as a result of truncating mutations of this gene. Clinical manifestations of CACP include congenital or early-onset camptodactyly, noninflammatory arthropathy with synovial hyperplasia and progressive coxa vara deformity, all symptoms related with the cartilage's physiology (Choi et al., 2004).

All these studies show that PRG4 is closely related to morphogenesis, maintenance and functioning of the AC as well as defects in its expression are related to the development of OA. That is why in recent years has aroused interest in the field of *in vitro* studies as a potential aim in developing AC regenerative strategies.

#### *4.1.2. PRG4: An in vitro, in vivo and clinical marker of chondrogenesis*

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

**4. The recapitulation of AC morphogenesis through MSCs differentiation**

In spite of therapeutic strategies mentioned, which offer the patient a temporary relief of symptoms, they do not resolved, in the medium or long term, the disease that affects the joint. In most cases, fibrocartilage is generated that does not provide the necessary structural integrity and this often results in the subsequent replacement of the damaged joint (Mahmou‐

The main problem with these strategies is that only tries to restore the biomechanical charac‐ teristics of tissue but not its physiology. That is why the goal of current research in AC regeneration with MSCs is moving to methods that attempt to recapitulate the morphogenesis of the tissue. The challenge is to develop strategies that will cover the widest possible range of intervening factors including: the dynamics of genes and proteins that control and partici‐ pate in the chondrogenic process, their spatiotemporal patterns of expression, variations in culture conditions, biomaterials functionalized with effector molecules inducers of chondro‐ genesis and the inclusion of differentiation enhancers using genetic engineering and others.

As previously mentioned, the AC's ECM is composed mainly of collagen type II fibers and proteoglycans with strong negative charges, aggrecan being the most abundant of them (Han et al, 2011). That is why the detection of both proteins and their corresponding genes has been classicallyusedasmarkersof chondrogenesis innumerous studies *invitro* and*invivo*.However, most modern regenerative strategies should aim at obtaining a more complete and organized ECM that achieves the greatest similarity with the ECM on the original AC. With this objec‐ tive, have been identified dozens of genes that play a fundamental role in the formation and maintenance of AC, which are potential targets for research in the design of regenerative

A good example is the proteoglycan 4 (PRG4), also known as "superficial zone protein" (SZP) and "lubricin". It is a proteoglycan specifically synthesized by chondrocytes located at the

**4.1. Novel genes and protein: The path to recapitulating AC morphogenesis**

strategies (Quintana et al., 2009; Bobick et al., 2009; Mahmoudifar and Doran, 2012).

*4.1.1. Proteoglycan 4 (PRG4): Biological function and its relationship to OA*

arises.

difar and Doran, 2012).

166 Regenerative Medicine and Tissue Engineering

Several studies show that this protein participates in the chondrogenic process and that its expression can be induced on *in vitro* chondrogenesis experiments. Recently, has been found an increase in *PRG4* gene expression in cells derived from infrapattelar fat pad (IFP) in response to treatment with TGF-β1 and BMP-7, demonstrating that the *in vitro* induction of this gene expression is possible. Furthermore, it served to demonstrate the suitability of the source cell used (Lee et al., 2008). However, the same research group in a similar characteristics experi‐ ment but using human embryonic stem cells (hESC) was able to induce chondrogenesis but failed inducing PRG4 expression, concluding that the induction conditions should be opti‐ mized. PRG4 use as a marker allowed the discrimination of both experiments (Nakagawa et al., 2009). It has also been observed that in rat skeletal muscle-derived mesenchymal stem/ progenitor cells (MDMSCs) treated with TGF-β1 and BMP-7, PRG4 increased in a timedependent manner on days 3, 7 and 10. As early as day 3, there was a three-fold increase of the PRG4 detected by ELISA analysis. This was confirmed by immunochemical localization of PRG4 as early as day 3 after treatment with TGF-β1. Even, the mRNA expression of PRG4 was enhanced by the two factors, along or in combination. This work demonstrates that is possible induce *in vitro* not only the increased of PRG4 gene expression, but also the accumulation of the protein in the medium (Andrades et al., 2012).

In clinical setting, there are also studies related with PRG4. Patients with OA tend to form small cartilaginous deposits in the exposed subchondral bone. The histological study of aggregates of patients undergoing total knee replacement, reveals that these aggregates were fibrocartilaginous, positive staining for glucosaminoglycan Safranin-O and type II collagen expression but, more interestingly, of PRG4. In those aggregates embedded in the bone, the staining was positive for the entire surface while in which protruded to the surface, the PRG4 was detected only in the edge surface as would be observed in normal cartilage. This *in vivo* observation is an excellent example of the cell's genetic response to the environment. Osteo‐ chondroprecursors contact with the synovial fluid and physicochemical stimuli inherent to the articular surface is able to modify the spatial distribution patterns of PRG4 expression and thus, the tissue architecture. These results are an invitation to test culture conditions that attempt to emulate not only the biochemical environment, but also mimic the biophysical characteristics of the physiological niche (Zhang et al., 2007) (Fig. 6).

**Figure 6.** A) Probe pointing to a white spot on the exposed bone surface of an osteoarthritic femoral condyle ob‐ tained at the time of total knee arthroplasty. B) Immunohistochemistry for PRG4 to subsurface chondrocyte aggregate in subchondral bone staining fully for PRG4; and C) fibrocartilaginous deposit protruding through the joint surface containing PRG4 in a zone just below the surface. From the same authors, published by Journal of orthopaedic re‐ *search: official publication of the Orthopaedic Research Society 25(7): 873–883. Copyright 2007*

It has also been demonstrated the possibility of reversing the decline in the expression of PRG4 in cartilage chondrocytes culture from OA patients. In this study, cartilage explants were obtained from healthy and OA patients and performed monolayer cultures and encapsulated in poly (ethylene glycol) diacrylate scaffold (PEG-DA). The OA cartilage explants have weaker inmunolabeling of PRG4 than healthy cartilage explants. However, the difference was reduced significantly between these 2 samples when were cultured in PEG-DA hydrogels. OA chon‐ drocytes regain the ability to express PRG4 at levels virtually identical to those obtained in chondrocytes from normal cartilage demonstrating the importance of culture conditions in the condrogenic induction (Musumeci et al., 2011).

All these evidences indicate that the inclusion of proteins as PRG4 in experimental designs offers advantages such as:


**3.** The ability to understand more deeply the dynamics of diseases that affect the AC like OA, in order to raise regenerative strategies that offer to patients, medium and long term solutions.

#### **4.2. Genetic engineering: Enhance of chondrogenic potencial beyond biochemical signals and functionalized scaffolds**

#### *4.2.1. SOX9: The key regulator of the chondrogenic process*

staining was positive for the entire surface while in which protruded to the surface, the PRG4 was detected only in the edge surface as would be observed in normal cartilage. This *in vivo* observation is an excellent example of the cell's genetic response to the environment. Osteo‐ chondroprecursors contact with the synovial fluid and physicochemical stimuli inherent to the articular surface is able to modify the spatial distribution patterns of PRG4 expression and thus, the tissue architecture. These results are an invitation to test culture conditions that attempt to emulate not only the biochemical environment, but also mimic the biophysical

**Figure 6.** A) Probe pointing to a white spot on the exposed bone surface of an osteoarthritic femoral condyle ob‐ tained at the time of total knee arthroplasty. B) Immunohistochemistry for PRG4 to subsurface chondrocyte aggregate in subchondral bone staining fully for PRG4; and C) fibrocartilaginous deposit protruding through the joint surface containing PRG4 in a zone just below the surface. From the same authors, published by Journal of orthopaedic re‐

It has also been demonstrated the possibility of reversing the decline in the expression of PRG4 in cartilage chondrocytes culture from OA patients. In this study, cartilage explants were obtained from healthy and OA patients and performed monolayer cultures and encapsulated in poly (ethylene glycol) diacrylate scaffold (PEG-DA). The OA cartilage explants have weaker inmunolabeling of PRG4 than healthy cartilage explants. However, the difference was reduced significantly between these 2 samples when were cultured in PEG-DA hydrogels. OA chon‐ drocytes regain the ability to express PRG4 at levels virtually identical to those obtained in chondrocytes from normal cartilage demonstrating the importance of culture conditions in the

All these evidences indicate that the inclusion of proteins as PRG4 in experimental designs

**1.** The inclusion of more highly specific markers that allow a better understanding of chondrogenic potential of cell sources, bioactive scaffolds and treatments applied.

**2.** Implant design that better mimic the characteristics of the AC and try to emulate not only

*search: official publication of the Orthopaedic Research Society 25(7): 873–883. Copyright 2007*

condrogenic induction (Musumeci et al., 2011).

the composition but also tissue architecture.

offers advantages such as:

characteristics of the physiological niche (Zhang et al., 2007) (Fig. 6).

168 Regenerative Medicine and Tissue Engineering

Given the low regenerative capacity of the AC, it is vital find ways to increase the chondrogenic potential of bioimplants designed. Today, not only can enhance the biochem‐ ical environment biomaterials and implants, but genetic engineering can increase the potential of MSCs used, changing their gene expression patterns from "inside". For this, it is important to consider transcription factors and their intracellular signaling cascades. Perhaps the most studied of these factors is SOX9, considered the key regulator of the chondrogenic process (Bi et al., 1999). The expression of SOX9 is upregulated by mem‐ bers of the FGFs, TGF-βs and BMPs, all of them widely used chondroinducers. In turn, SOX9 is responsible for regulating SOX5, SOX6 and activating the expression of collagens type II, VI, IX and XI, the proteoglycans Aggrecan, Byglican and Perlecan and important binding proteins as COMP (Quintana et al., 2009) (Fig. 7).

On the other hand, during skeletogenesis, SOX9 is responsible for the osteochondroprogeni‐ tors differentiation into chondroblasts and not into osteoblasts to direct and indirect repression of RUNX2 (the main regulator of osteogenic differentiation) favoring the endochondral ossification (Zhou et al., 2006).

#### *4.2.2. Transfection of SOX9 as an activator of chodrogenesis*

The application of these regulatory genes on regenerative design strategies could be useful to increase both specificity and efficiency of the bioimplants. This opens the possibility of using not only functionalized biocompatible scaffolds, but also cells previously treated to have a higher chondrogenic potential. In a recent study, human MSCs are transfected with a nonviral vector plasmid complexed with SOX9 cDNA in order to induce chondrogene‐ sis. Micromass culture and transplantation into nude mice of control and transfected cells were made. Both procedures showed increased levels of mRNA for COL2A1, Aggrecan and COMP; increased GAG content; alcian blue staining positive and detection of type II collagen and Aggrecan by immunofluorescence, all of the cells transfected with respect to control. Similar results were achieved using a viral vector transfection of SOX9 (Fig. 8). Additionally, this group demonstrated that transfection of the gene, in addition to induc‐ ing chondrogenesis, reduces the levels of markers of hypertrophy, osteogenesis and adipogenesis, thereby inhibiting the possibility that the human MSCs to differentiate into these mesenchymal lineages (Venkatesan et al., 2012).

**Figure 7.** Upstream and downstream regulation of Sox9. From the same authors, published by Tissue engineering. Part B, Reviews 15(1): 29–41. Copyright 2009

**Figure 8.** Schematic diagram of SOX9 gene transfection using a modified and non-modified biodegradable nanopar‐ ticles, an example of Non-viral transfection. During hMSCs transfection, nanoparticles interact with the negatively charged lipid bilayers and are influxed into endosomes and destabilized, resulting in the release of the transfected genes into the cytosol. *From the same authors, published by Biomaterials 32(1): 268–278. Copyright 2011*

The possibility of genetically engineering the different types of MSCs used in cartilage regeneration is a promising tool for increasing chondrogenic capacity and, consequently, improving future regeneration bioimplants. A deepest and detailed knowledge of gene regulation involved in the process and their possible clinical utilities will lead the way of successful recapitulation of AC morphogenesis through MSCs differentiation.
