Three-Dimensional Printing of Click Functionalized, Peptide Patterned Scaffolds for Osteochondral Tissue Engineering

Abstract

Osteochondral repair remains a significant clinical challenge due to the multiple tissue phenotypes and complex biochemical milieu in the osteochondral unit. To repair osteochondral defects, it is necessary to mimic the gradation between bone and cartilage, which requires spatial patterning of multiple tissue-specific cues. To address this need, we have developed a facile system for the conjugation and patterning of tissue-specific peptides by melt extrusion of peptide-functionalized poly(ε-caprolactone) (PCL). In this study, alkyne-terminated PCL was conjugated to tissue-specific peptides via a mild, aqueous, and Ru(II)-catalyzed click reaction. The PCL-peptide composites were then 3D printed by multimaterial segmented printing to generate user-defined patterning of tissue-specific peptides. To confirm the bioactivity of 3D printed PCL-peptide composites, bone- and cartilage-specific scaffolds were seeded with mesenchymal stem cells and assessed for deposition of tissue-specific extracellular matrix in vitro. PCL-peptide scaffolds successfully promoted osteogenic and chondrogenic matrix deposition, with effects dependent on the identity of conjugated peptide.

Keywords: 3D printing; bioconjugation; extrusion; osteochondral; patterning; scaffold.

Figure 1:

Click conjugation and three-dimensional printing of cartilage- and bone-specific composites. The tissue-specific polymers can be spatially patterned by multimaterial segmented printing.

Figure 2:

NMR characterization of PCL-alkyne and PCL-peptide composites, with (a) PCL-peptide conjugation scheme, (b) overall comparison of PCL-alkyne and PC-BMPm spectra, and (c) area of interest containing alkyne-adjacent peak and histidine side chain peaks for PCL-alkyne and PCL-peptide composites.

Figure 3:

Layer-by-layer images of melt extruded (a) PCL-alkyne, (b) PCL-BMPm, (c) PCL-GHK, and (d) PCL-NC scaffolds. Scale bar represents 1 mm. (e) Physical parameters of printed scaffolds, as measured by μCT. All data are reported as means ± standard deviation for a sample size of n=3.

Figure 4:

User-defined lateral patterning of fluorescently tagged PCL-peptide composites. (a) Heterogeneous design of construct layers, (b) image of printed construct, and (c) fluorescent image of printed construct. All scale bars represent 5 mm.

Figure 5:

Assessment of cellularity and tissue-specific matrix deposition in PCL-peptide scaffolds cultured for 0-28 days. (a) DNA content, representing cellularity. (b-c) Bone-specific calcium content, normalized to cellular content and to scaffold mass. (d-e) Cartilage-specific sulfated glycosaminoglycan (sGAG) content, normalized to cellular content and to scaffold mass. All data are reported as means ± standard deviation for a sample size of n=3. * indicates statistical significance compared to a peptide-free PCL-alkyne control in the same medium formulation and same timepoint, # indicates significance compared to the same scaffold material and medium formulation at Day 0, representing progression over time.