**3.3 Bicomponent scaffolds made out of PCL and self-assembling peptide hydrogels**

 Self-assembling peptide hydrogels provide a soft and permissive microenvironment, allowing cells to migrate, extend cellular processes and contact with other cells. Nevertheless, the use of soft hydrogels for CTE can be challenging due to its low stiffness. One approach to address this issue is the use of composite scaffolds, comprising a microscale component to increase mechanical properties and a hydrogel component (of nanoscale dimension) to promote chondrogenesis. Woven 3D poly(ε-caprolactone) (PCL) resemble native cartilage mechanical properties and, due to its high wettability, can be infiltrated with a hydrogel matrix, such as fibrin, alginate, and poly-acrylamide [43–45]. In this study, we developed a unique composite scaffold by infiltrating a 3D woven microfiber poly(ε-caprolactone) scaffold with the RAD16-I self-assembling peptide nanofiber to obtain a multi-scale functional cartilage-like tissue. The chondrogenic capacity of this new bicomponent was evaluated with expanded dedifferentiated human articular chondrocytes.

The high wettability properties of the PCL scaffold (**Figure 6A**) allowed to easily introduce the cells suspended in the RAD16-I peptide solution between the

#### **Figure 6.**

*SEM characterization of PCL/RAD and hACh constructs. (A) Water (left) and 0.5% RAD16-I solution (right) contact angle. The liquid was totally absorbed by the PCL scaffold (contact angle << 90°), indicating high wettability. (B) Surface view of PCL and PCL/RAD structure by SEM. 0.5% RAD16-I was lyophilized within the PCL scaffold. (C) hACh at week 4 of culture in PCL, PCL/RAD and RAD scaffolds. hACh were seeded in each scaffold and cultured in expansion, control and chondrogenic medium. Two images per condition are shown. Adapted from Recha-Sancho et al. [46].* 

interweaving fibers of PCL scaffold (**Figure 6B**, left). Areas of RAD16-I peptide deposition could be observed within the organized woven morphology of the fiber scaffold (**Figure 6B**, right). Thus, cells were seeded in the composite PCL/RAD and in the two scaffolds independently, PCL and RAD, and maintained for 4 weeks in expansion, chondrogenic and control medium. 3D constructs were analyzed for morphology, gene and protein expression, proteoglycan synthesis and mechanical properties.

In order to evaluate cell morphology and their interaction with the scaffolds, SEM images of hACh cultured in PCL, PCL/RAD and RAD 3D scaffolds in expansion, control and chondrogenic medium were taken at week 4 of culture (**Figure 6C**). hACh seeded in PCL scaffolds looked elongated and growing on the surface of PCL fibers. Interestingly, more fibers were detected under chondrogenic induction, probably due to an increase in extracellular matrix components production by the cells. In PCL/RAD constructs, cells seemed to be attached to the PCL fibers, with a more spherical morphology than in PCL scaffold alone, while hACh in RAD scaffolds presented in general a spherical shape.

Chondrogenic and hypertrophic markers were studied at gene and protein level at week 4 of culture in 3D scaffolds and compared to 2D cultures. *COL1* was downregulated or maintained at 2D culture levels under expansion medium, while it increased in all 3D constructs under chondrogenic conditions (**Figure 7A**). At protein level a single band (~220 kDa) was obtained for COL1 in 2D culture, while different bands of lower molecular weight (ranging from 180 to 130 kDa) were observed in 3D cultures of PCL/RAD and RAD (in all medium tested) and PCL in chondrogenic conditions (**Figure 7B**).

 The expression of *COL2* was only increased in PCL/RAD and PCL scaffolds under chondrogenic induction, however, significant differences were only detected

#### **Figure 7.**

*Chondrogenic capacity of dedifferentiated hACh in PCL, PCL/RAD and RAD scaffolds. hACh were seeded in each scaffold, 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. 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). (B) Protein expression characterization of hACh cultured in PCL, PCL/RAD and RAD scaffolds 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 and in 2D monolayer. Actin expression was used as an internal control. Samples were prepared in triplicate. (C) Toluidine blue staining of hACh 3D PCL, PCL/RAD and RAD constructs cultured in expansion, control and chondrogenic medium. Proteoglycan synthesis was qualitatively assessed by toluidine blue staining. Adapted from Recha-Sancho et al. [46].* 

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

 for RAD scaffold (**Figure 7A**). At protein level, COL2 was only detected in PCL/ RAD and PCL scaffolds under chondrogenic medium (**Figure 7B**). *SOX9* was downregulated in PCL scaffolds in both culture medium and PCL/RAD in expansion medium. Nevertheless, it was maintained similar to 2D levels in PCL/RAD composites under chondrogenic induction and in RAD scaffold (**Figure 7A**). Aggrecan (*ACAN*) gene expression was downregulated in all scaffolds under expansion medium and upregulated in all scaffolds under chondrogenic medium, even though no differences were detected relative to 2D cultures (**Figure 7A**). Hypertrophic markers *COL10* and *RUNX2* were upregulated in some constructs respect to baseline. However, no significant increase for *COL10* was detected in RAD and PCL/ RAD constructs under chondrogenic medium (**Figure 7A**). At protein level, COL10 was detected in all samples (**Figure 7B**).

The production of sulfated glycosaminoglycans was qualitatively assessed by toluidine blue staining. Constructs under chondrogenic medium were the most strongly stained compared to expansion and control medium (**Figure 7C**).

 Mechanical properties of the scaffolds alone and hACh 3D constructs were assessed by dynamic mechanical analysis (DMA) at week 4 of culture, and compared to chicken and calf articular cartilage (**Figure 8**). The elastic component (G′, storage modulus) of scaffolds and 3D cultures was significantly lower than values of chicken and calf cartilage. Regarding the viscous component (G″, loss modulus), 3D constructs differed from calf native cartilage, while only PCL cellular scaffolds presented differences with chicken cartilage. All samples presented G′ values higher than G″ values, meaning that the material was more elastic than viscous. Because the complex modulus (G\*) is the sum of both components, G\* basically corresponds to the elastic component in this case and it presented the same pattern as the storage modulus (G′). Concerning tan(delta), which is the full mechanical response of the material, the scaffolds and cell constructs were closely related to both native cartilages, with exception of RAD constructs in chondrogenic medium, which presented differences with calf cartilage. Moreover, differences were observed between PCL/RAD and RAD constructs under the same medium. The combination of PCL scaffold and RAD hydrogel changed their viscoelastic nature after 4 weeks of culture with hACh, since tan(delta) values of the composite were increased compared to RAD scaffolds alone. This effect was not observed between composites PCL/RAD and PCL scaffold alone.

In the present study we report the chondrogenic capacity of dedifferentiated hACh in a composite scaffold comprising a microscale woven 3D poly (ε-caprolactone) and the peptide nanofiber scaffold RAD16-I. PCL scaffold resembles native cartilage mechanical properties while the RAD16-I hydrogel provides a soft and permissive 3D environment. The expression of chondrogenic markers such

#### **Figure 8.**

*Mechanical characterization of scaffolds alone and 3D constructs cultured for 4 weeks in expansion, control and chondrogenic medium compared to chicken and calf articular cartilage. hACh cultured in PCL, PCL/ RAD and RAD 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 et al. [46].* 

as *COL2* and *ACAN* was increased in the presence of RAD16-I peptide (in the composite and alone) compared to 2D cultures (**Figure 7A**). At protein level, different band patterns were detected for COL1, fact that suggests a protein maturation process. Specifically, the scaffolds PCL/RAD and RAD alone under chondrogenic induction, expressed higher levels of the mature COL1, as evidenced by the intensity of the 130 kDa band. Moreover, COL2 was only detected in PCL/RAD and RAD scaffolds under chondrogenic medium, suggesting that the expression of this cartilage-specific protein was due to the presence of RAD16-I hydrogel (**Figure 7B**). GAG production and accumulation was confirmed by toluidine blue staining in constructs under chondrogenic medium (**Figure 7C**). Finally, mechanical characterization showed that at the end of culture, all constructs had a viscoelastic nature (tan delta) similar to native articular cartilage, even though G′ values differed several folds from native cartilage (**Figure 8**). In resume, is clear that the combination of biomaterials to obtain a multi-dimensional composite (microfiber and nanofiber scales) is essential to acquire the best culture conditions for the cells to undergo cartilage lineage differentiation.
