RESUMO
The regeneration of the osteochondral unit represents a challenge due to the distinct cartilage and bone phases. Current strategies focus on the development of multiphasic scaffolds that recapitulate features of this complex unit and promote the differentiation of implanted bone-marrow derived stem cells (BMSCs). In doing so, challenges remain from the loss of stemness during in vitro expansion of the cells and the low control over stem cell activity at the interface with scaffolds in vitro and in vivo. Here, this work scaffolds inspired by the bone marrow niche that can recapitulate the natural healing process after injury. The construct comprises an internal depot of quiescent BMSCs, mimicking the bone marrow cavity, and an electrospun (ESP) capsule that "activates" the cells to migrate into an outer "differentiation-inducing" 3D printed unit functionalized with TGF-ß and BMP-2 peptides. In vitro, niche-inspired scaffolds retained a depot of nonproliferative cells capable of migrating and proliferating through the ESP capsule. Invasion of the 3D printed cavity results in location-specific cell differentiation, mineralization, secretion of alkaline phosphatase (ALP) and glycosaminoglycans (GAGs), and genetic upregulation of collagen II and collagen I. In vivo, niche-inspired scaffolds are biocompatible, promoted tissue formation in rat subcutaneous models, and regeneration of the osteochondral unit in rabbit models.
RESUMO
Melt extrusion-based additive manufacturing (AM) is often used to fabricate scaffolds for osteochondral (OC) regeneration. However, there are two shortcomings associated with this scaffold manufacturing technique for engineering of tissue interfaces: (a) most polymers used in the processing are bioinert, and (b) AM scaffolds often contain discrete (material) gradients accompanied with mechanically weak interfaces. The inability to mimic the gradual transition from cartilage to bone in OC tissue leads to poor scaffold performance and even failure. We hypothesized that introducing peptide gradients on the surface could gradually guide human mesenchymal stromal cell (hMSC) differentiation, from a chondrogenic towards on osteogenic phenotype. To work towards this goal, we initially manufactured poly(ϵ-caprolactone)-azide (PCLA) and PCL-maleimide (PCLM) scaffolds. The surface exposed click-type functional groups, with a surface concentration in the 102pmol cm-2regime, were used to introduce bone morphogenic protein-2 or transforming growth factor-beta binding peptide sequences to drive hMSC differentiation towards osteogenic or chondrogenic phenotypes, respectively. After 3 weeks of culture in chondrogenic medium, we observed differentiation towards hypertrophic chondrogenic phenotypes with expression of characteristic markers such as collagen X. In osteogenic medium, we observed the upregulation of mineralization markers. In basic media, the chondro-peptide displayed a minor effect on chondrogenesis, whereas the osteo-peptide did not affect osteogenesis. In a subcutaneous rat model, we observed a minimal foreign body response to the constructs, indicating biocompatibility. As proof-of-concept, we finally used a novel AM technology to showcase its potential to create continuous polymer gradients (PCLA and PCLM) across scaffolds. These scaffolds did not display delamination and were mechanically stronger compared to discrete gradient scaffolds. Due to the versatility of the orthogonal chemistry applied, this approach provides a general strategy for the field; we could anchor other tissue specific cues on the clickable groups, making these gradient scaffolds interesting for multiple interfacial tissue applications.