**3. Representative results**

Autologous chondrocytes are one of the most attractive cell types for CTE, due to their intrinsic properties regarding cell function, since they are found in the native cartilage. Chondrocytes are characterized by a rounded morphology, the production of tissue-specific ECM components such as collagen type I and II and glycosaminoglycans (GAGs). One of the main challenges in CTE is to obtain enough cell mass to develop a tissue construct with the desirable biological and biomechanical properties. Particularly, articular chondrocytes are obtained by invasive techniques and cell number in patient biopsies is limited. Therefore, after isolation, chondrocytes need to be expanded in 2D monolayer [1]. The expansion process leads to a rapid downregulation of chondrogenic markers, such as Collagen type I (COL1) collagen type II (COL2) and Aggrecan (ACAN) [8, 9]. Moreover, the use of extensively passaged cells leads to some degree of hypertrophy, decreased biochemical content and compromised mechanical properties [1], which is not a good indication for cartilage substitute applications.

Mesenchymal stem cells (MSCs) have generated great interest as an alternative cell source to autologous chondrocytes. MSCs are pluripotent cells with a high proliferative capacity that can be differentiated, under the appropriate microenvironment, to numerous cell lineages, such as osteogenic, adipogenic and chondrogenic [35]. MSCs can be isolated from bone marrow, adipose tissue and other sources. In particular, the adipose tissue provides and abundant reservoir of mesenchymal stem cells (adipose-derived stem cells, ADSC), which can be obtained by non-invasive surgical techniques. ADSC can undergo chondrogenic commitment in the presence of TGF-β, ascorbate, and dexamethasone combined with a 3D culture environment [35].

Three-dimensional scaffold-based cell cultures are currently used in CTE to reestablish chondrogenic phenotype of dedifferentiated chondrocytes, since they mimic more closely the natural tissue environment. On the other hand, differentiation of ADSC to cartilage-like tissue has been achieved in various 3D scaffold systems such as alginate [36], agarose [37] and collagen [38]. We report here the development of new bicomponent scaffolds based on the self-assembling peptide RAD16-I, for guiding chondrogenic differentiation of both adipose-derived stem cells (ADSC) and expanded dedifferentiated human articular chondrocytes (hAChs).

## **3.1 Bicomponent scaffolds made out of heparin/self-assembling peptide hydrogels**

In this section, we report the development of a nanofiber scaffold with growth factor binding affinity. The strategy consisted of adding heparin moieties to the RAD16-I peptide scaffold by mixing the two components, forming a stable composite hydrogel scaffold with a natural capacity to retain HBD-containing growth factors. To evaluate the functionality of this approach for CTE applications, ADSC were cultured in the new bicomponent scaffold and induced to chondrogenic differentiation using TGFβ-1, l-ascorbic acid 2-phosphate and dexamethasone as inductors in serum-free media. 3D cultures were maintained for 4 weeks in chondrogenic or control medium, and analyzed for proteoglycan production, protein expression and mechanical properties.

 During ADSC culture in the peptide scaffold RAD16-I combined with increasing concentrations of heparin (RAD/Hep), constructs cultured under chondrogenic medium—unlike constructs under control medium—became highly stained with toluidine blue, indicating a significant production of proteoglycans (**Figure 2A**). This result correlated with the aggrecan (*ACAN*) gene expression, which was only detected in constructs under chondrogenic induction (**Figure 2B**). ADSC cultured within RAD/Hep composites also produced cartilage-specific ECM proteins, such as COL1, COL2 and COL10 (**Figure 2C**). Interestingly, a single band was obtained for COL1 in 2D culture, corresponding probably to a pro-collagen intermediate (approx. 220 kDa). Different bands (ranging from 130 to 180 kDa) were obtained for COL1 in 3D constructs under chondrogenic induction. Importantly, COL2 was only detected in 3D chondro-induced cultures.

Moreover, mechanical characterization was performed over 3D chondro-induced constructs. 3D constructs, presented a storage modulus (G′) in the same order of magnitude to chicken or calf articular cartilage, but the full mechanical response of the constructs was different from native cartilage as evidenced by tan(delta) (**Figure 2D**).

## **3.2 Bicomponent scaffolds made out of chondroitin sulfate or decorin and selfassembling peptide hydrogels**

The next strategy was based on mimicking the native cartilage ECM by adding chondroitin sulfate or decorin molecules to the nanofiber scaffold, generating thus *Cartilage Tissue Engineering Using Self-Assembling Peptides Composite Scaffolds DOI: http://dx.doi.org/10.5772/intechopen.83716* 

#### **Figure 2.**

*Chondrogenic capacity of ADSC in RAD/heparin composite scaffold. ADSC were encapsulated within the RAD16-I peptide scaffold combined with increasing concentrations of heparin and cultured for 4 weeks under control and chondrogenic medium. (A) Toluidine blue staining of 3D ADSC constructs cultured under control and chondrogenic medium. 3D construct view scale bars = 500 μm and section close up scale bars = 100 μm. (B) Aggrecan gene expression levels of chondro-induced ADSC. Constructs cultured with control medium did not express aggrecan after 4 weeks of culture. Ct values relative to ribosomal protein L22 (RPL22) were obtained and reported as fold increase (ΔΔCt) relative to 2D cultures. (C) Protein expression characterization of ADSC cultured in RAD/Hep composites and in 2D monolayer. Western blot results of collagen type I, II and X when ADSC were maintained in control and chondrogenic medium in RAD16-I scaffold and RAD16-I/Hep composites. Actin expression was used as an internal control. (D) Mechanical characterization of 3D constructs cultured for 4 weeks in chondrogenic medium compared to chicken and calf articular cartilage. ADSCs cultured with RAD16-I and RAD/Hep composite scaffolds were analyzed for storage modulus (G′, A), loss modulus (G″, B), complex modulus (G\*, C) and tan(delta). Significant differences are indicated as \* for p < 0.05, \*\* for p < 0.01, and \*\*\* for p < 0.001, one-way ANOVA, N = 2 n = 3). Adapted from Fernández-Muiños et al. [25].* 

 chondro-favorable biochemical cues in the 3D environment. Previous work has evaluated the influence of CS to guide chondrogenesis in different hydrogel scaffolds such as chitosan [39], PEG [40], or collagen type I [41], but less is known about the ability of decorin to promote chondrogenesis commitment. In the present work, we studied the influence of both CS and decorin molecules on chondrogenesis in a nanometric 3D system. The capacity of these bicomponent scaffolds to foster chondrogenic differentiation was evaluated in two different scenarios: re-differentiation of expanded hAChs and induction of ADSC to chondrogenic commitment. Cells were seeded in RAD16-I/CS, RAD16-I/Dec and RAD16-I scaffold alone and maintained for 4 weeks in chondrogenic or control medium. Moreover, chondrocytes were also cultured in expansion medium, which contains GFs that could affect the fate of the 3D culture. 3D constructs were analyzed for morphology, gene and protein expression, proteoglycan synthesis and mechanical properties.

 SEM images were obtained at week 4 of culture to assess cell morphology and their interaction with each scaffold (**Figure 3**). Articular chondrocytes cultured in expansion medium possessed a spherical morphology with possible cell-matrix interactions and thorough ECM components. Nanofibers and putative matrix components were detected on the surface of constructs cultured in control medium. Moreover, grooves with visible fibers were observed on the surface of constructs cultured in chondrogenic medium, fact that suggested the presence of secreted matrix components. On the other hand, adipose-derived stem cells under chondrogenic induction looked elongated and anchored to the scaffold surface, while

#### **Figure 3.**

*SEM images of hACh and ADSC at week 4 of culture in RAD, RAD/CS and RAD/Dec scaffolds. Two images per condition are shown. Adapted from Recha-Sancho and Semino [42].* 

 nanofibers and possible ECM components synthesized by the cells were observed in control medium (**Figure 3**). No significant differences in cell morphology were detected between RAD, RAD/CS or RAD/Dec scaffolds in any cell type.

 Chondrogenic markers expression were studied at gene and protein level in hACh 3D constructs cultured in chondrogenic and expansion medium, and compared to their 2D counterparts. *COL1* was upregulated in all 3D scaffolds under chondrogenic medium and downregulated under expansion medium (**Figure 4A**). At protein level, COL1, was detected both in 2D monolayer and 3D constructs, but different band patterns were observed (**Figure 4B**). In 2D cultures, a single band was detected (approx. 220 kDa), generated probably by a pro-collagen intermediate. In 3D cultures, different bands of lower molecular weight (ranging from 130 to 180 kDa) were observed, but their intensity varied depending on the culture medium.

 Interestingly, *COL2* gene expression was only upregulated in RAD/CS and RAD/Dec composite scaffolds under chondrogenic medium. This result correlated with the expression of *SOX9*, a gene regulator of *COL2*, which was significantly upregulated in 3D constructs under chondrogenic induction (**Figure 4A**). At protein level, COL2 was only detected in 3D cultures under chondrogenic induction, fact that was consistent with the gene expression profile results (**Figure 4B**). *ACAN* gene expression was higher in constructs under chondrogenic medium than in constructs cultured under expansion medium (**Figure 4A**). No differences were detected in the gene expression of hypertrophic markers compared to 2D cultures, except in RAD16-I scaffold alone, where the expression of *COL10* was upregulated in expansion medium, and *RUNX2* in chondrogenic medium (**Figure 4A**). COL10 protein expression was observed in all conditions, including 2D, but more intense bands were detected in expansion and chondrogenic medium, compared to control (**Figure 4B**).

Toluidine blue staining was performed in hACh 3D constructs to qualitatively assess the production of GAGs. Constructs under chondrogenic induction became highly stained, indicating a significant production and accumulation of GAGs by the cells (**Figure 4C**). Constructs cultured under expansion medium showed less staining, while constructs under control medium became weakly stained.

*Cartilage Tissue Engineering Using Self-Assembling Peptides Composite Scaffolds DOI: http://dx.doi.org/10.5772/intechopen.83716* 

#### **Figure 4.**

*Chondrogenic capacity of dedifferentiated hACh in RAD/CS and RAD/Dec 3D composite scaffolds. hACh were encapsulated within the RAD16-I peptide scaffold combined with chondroitin sulfate and decorin, and cultured for 4 weeks under expansion, control and chondrogenic medium. (A) Gene expression levels of chondrogenic and hypertrophic markers. hACh were analyzed by qRT-PCR for collagen type I (COL1), collagen type II (COL2), SOX9, aggrecan (ACAN), collagen type X (COL10) and RUNX2. Ct values relative to ribosomal protein L22 (RPL22) were obtained and reported as the fold increase (ΔΔCt) relative to 2D cultures (B) protein expression characterization of hACh cultured in RAD, RAD/CS and RAD/Dec composites and in 2D monolayer. Western blot results of collagen type I (COL1), II (COL2) and X (COL10) when hACh were maintained in expansion, control and chondrogenic media in the different scaffolds (RAD, RAD/CS and RAD/Dec) and in 2D monolayer. Actin expression was used as an internal control. Samples were prepared in triplicate. (C) Toluidine blue staining of hACh 3D RAD, RAD/CS and RAD/Dec constructs cultured in expansion, control and chondrogenic medium. Proteoglycan synthesis was qualitatively assessed by toluidine blue staining. (D) Mechanical characterization of 3D constructs cultured for 4 weeks in chondrogenic medium compared to chicken and calf articular cartilage. hACh cultured with RAD16-I and RAD/CS and RAD/Dec composite scaffolds were analyzed for storage modulus (G′), loss modulus (G″), complex modulus (G\*) and tan(delta). Significant differences are indicated as \* for p < 0.05, \*\* for p < 0.01, and \*\*\* for p < 0.001, one-way ANOVA, N = 2 n = 3). Adapted from Recha-Sancho and Semino [42].* 

The mechanical properties of hACh 3D constructs cultured under chondrogenic medium were assessed at week 4 by dynamic mechanical analysis (DMA) and compared to calf and chicken articular cartilage (**Figure 4D**). hACh constructs exhibited lower storage modulus values (G′) than did the native cartilage samples. The viscous components (G″) and the complex modulus (G\*) displayed a more similar tendency to cartilage controls. Nevertheless, all samples presented G′ values higher than G″ values, indicating that the constructs were more elastic than viscous. Tan(delta) showed that 3D constructs were comparable to chicken cartilage but differed from calf cartilage.

 Chondrogenic and hypertrophic markers were studied in ADSC 3D constructs in the three scaffold types and compared to 2D monolayer culture. Results show that the gene expression of *COL1* was downregulated in 3D cultures. However, the expression of *COL2*, *SOX9* and *ACAN* was increased in 3D cultures compared to 2D. The expression levels of the hypertrophic markers *COL10* and *RUNX2* in 3D cultures were maintained at comparable levels to 2D culture (**Figure 5A**). At protein level, ADSC under chondrogenic induction produced cartilage-specific ECM proteins such as COL1, COL2 and COL10 (**Figure 5B**). As happened for hACh, one single band was obtained for *COL1* in 2D monolayer, while different bands of lower molecular weight were observed in 3D cultures. Interestingly, COL2 protein was only detected in 3D cultures.

Chondro-induced ADSC produced sulfated glycosaminoglycans, as reveals the intense staining by toluidine blue (**Figure 5C**, up). No calcium mineralization, an indicator of hypertrophy, was detected by Von Kossa staining (**Figure 5C**, down).

 The mechanical properties of ADSC cultured under chondrogenic conditions in RAD, RAD/CS and RAD/Dec were assessed by dynamic mechanical analysis (DMA) at week 4 (**Figure 5D**). The constructs presented a storage modulus (G′), viscous component (G″) and complex modulus (G\* ) closely related to chicken and calf cartilage. However, samples presented values of G′ much higher than G´´ so that the constructs were more elastic than viscous. Tan(delta) showed that the full mechanical response of the constructs was very similar to chicken cartilage but differed from calf cartilage.

In the present work, we aimed to induce chondrogenesis differentiation of both expanded hACh and ADSC in 3D bicomponent scaffolds made out of chondroitin sulfate or decorin and self-assembling peptide hydrogels. The expression of chondrogenic markers such as *COL2*, *SOX9* and *ACAN* was increased in both cell types compared to monolayer cultures (**Figures 4A** and **5A**). At protein level, western blot results showed a possible COL1 maturation process in 3D cultures of both cell types compared to 2D protein expression. In particular, the final mature COL1 product corresponds to the lower molecular weight band (130 kDa), which was absent in 2D cultures but predominant in constructs under chondrogenic medium (**Figures 4B** and 5**B**). Importantly,

#### **Figure 5.**

*Chondrogenic capacity of ADSC in RAD/CS and RAD/Dec 3D composite scaffolds. ADSC were encapsulated within the RAD, RAD/CS and RAD/Dec composite scaffolds and cultured for 4 weeks under control and chondrogenic medium. (A) Gene expression levels of chondrogenic and hypertrophic markers. ADSC were analyzed by qRT-PCR for collagen type I (COL1), collagen type II (COL2), SOX9, aggrecan (ACAN), collagen type X (COL10) and RUNX2. Ct values relative to ribosomal protein L22 (RPL22) were obtained and reported as the fold increase (ΔΔCt) relative to 2D cultures. (B) Protein expression characterization of ADSC cultured in RAD, RAD/CS and RAD/Dec composites and in 2D monolayer. Western blot results of collagen type I (COL1), II (COL2) and X (COL10) when ADSC were cultured under control and chondrogenic medium in the different scaffold types. Actin expression was used as an internal control. (C) Toluidine blue and Von Kossa staining of 3D ADSC constructs under chondrogenic induction. Proteoglycan synthesis was qualitatively assessed by toluidine blue staining (up) and calcium mineralization by Von Kossa staining (down). (D) Mechanical characterization of 3D constructs cultured for 4 weeks in chondrogenic medium compared to chicken and calf articular cartilage. ADSCs cultured with RAD16-I and RAD/CS and RAD/Dec composite scaffolds were analyzed for storage modulus (G′, A), loss modulus (G″, B), complex modulus (G\*, C) and tan(delta). Significant differences are indicated as \* for p < 0.05, \*\* for p < 0.01, and \*\*\* for p < 0.001, one-way ANOVA, N = 2 n = 3). Adapted from Recha-Sancho and Semino [42].* 

### *Cartilage Tissue Engineering Using Self-Assembling Peptides Composite Scaffolds DOI: http://dx.doi.org/10.5772/intechopen.83716*

COL2 expression was only detected in 3D cultures under chondrogenic induction. Moreover, GAG production and accumulation was confirmed by toluidine blue staining (**Figures 4C** and 5**C**). Altogether, these results indicate the synergistic effect of the 3D culture system and the chemical inducers present in the chondrogenic medium in activating signaling pathways essentials for chondrogenic commitment, in terms of production of proteins and GAG components of the ECM. Finally, mechanical characterization showed that the viscoelastic behavior of chondro-induced ADSC constructs was more similar to native cartilage than hACh constructs (**Figures 4D** and **5D**). In resume, results until this section clearly indicate the chondro-inductive capacity of the modified scaffold which reinforce the development of biomimetic microenvironments to promote better tissue engineered cartilage substitutes.
