A highly porous type II collagen containing scaffold for the treatment of cartilage defects enhances MSC chondrogenesis and early cartilaginous matrix deposition.

A major challenge in cartilage tissue engineering (TE) is the development of instructive and biomimetic scaffolds capable of driving effective mesenchymal stem cell (MSC) chondrogenic differentiation and robust de novo matrix formation. Type I collagen-based scaffolds are one of the most commonly selected materials given collagen's intrinsic ability to act as an instructive and active biomaterial. However, the chondrogenic potential of these scaffolds does not offer significant improvement over traditional treatments. We propose that taking a biomimetic approach to scaffold development might lead to an improved outcome for enhanced cartilage repair. Therefore, this study aimed to develop innovative type II collagen (CII)-containing scaffolds for enhanced cartilage repair, by incorporating CII and/or hyaluronic acid (HyA) into a type I collagen (CI) framework. Moreover, focus was placed on understanding the potential synergistic effects played by CII in combination with HyA, in terms of MSC chondrogenesis and cartilage-like formation, when both molecules are incorporated into scaffold biomaterials. The newly developed CII-containing scaffold exhibited a highly porous interconnected structure with 99% porosity and similar mechanical properties to previously optimised collagen-based scaffolds. Although all scaffold variants sustained early cartilaginous matrix deposition, the CII-containing scaffolds in the presence of HyA performed best, offering enhanced deposition and distribution of sulphated glycosaminoglycans (sGAG) in vitro by day 28. Taken together, the combination of CII and HyA resulted in the development of a biomimetic scaffold with improved chondrogenic benefits. These simple "off-the-shelf" implants hold great promise to direct enhanced tissue regeneration for the treatment of focal cartilage defects.

The lubricating effect of iPS-reprogrammed fibroblasts on collagen-GAG scaffolds for cartilage repair applications.

Tissue engineering products, like collagen-glycosaminoglycan scaffolds, have been successfully applied to chondrogenic defects. Inducible Pluripotent Stem cell (iPS) technology allows reprograming of somatic cells into an embryonic-like state, allowing for redifferentiation. We postulated that a fibroblast cell line (BJ cells - 'pre-iPSF') cycled through iPS reprogramming and redifferentiated into fibroblasts (post-iPSF) could lubricate collagen-glycosaminoglycan scaffolds; fibroblasts are known to produce lubricating molecules (e.g., lubricin) in the synovium. Herein, we quantified the coefficient of friction (CoF) of collagen-glycosaminoglycan scaffolds seeded with post-iPSF; tested whether cell-free scaffolds made of post-iPSF derived extracellular matrix had reduced friction vs. pre-iPSF; and assessed lubricin quantity as a possible protein responsible for lubrication. Post-iPSF seeded CG had 6- to 10-fold lower CoF versus pre-iPSF. Scaffolds consisting of a collagen and pre-/post-iPSF extracellular matrix blend outperformed these cell-seeded scaffolds (~5-fold lower CoF), yielding excellent CoF values close to synovial fluid. Staining revealed an increased presence of lubricin within post-iPSF scaffolds (confirmed by western blotting) and on the surface of iPSF-seeded collagen-glycosaminoglycan scaffolds. Interestingly, when primary cells from patient biopsy-derived fibroblasts were used, iPS reprogramming did not further reduce the already low CoF of these cells and no lubricin expression was found. We conclude that iPS reprogramming activates lubricating properties in iPS-derived cells in a source cell-specific manner. Additionally, lubricin appears to play a lubricating role, yet other proteins also contribute to lubrication. This work constitutes an important step for understanding post-iPSF lubrication of scaffolds and its potential for cartilage tissue engineering.

The Incorporation of Marine Coral Microparticles into Collagen-Based Scaffolds Promotes Osteogenesis of Human Mesenchymal Stromal Cells via Calcium Ion Signalling.

Composite biomaterial scaffolds consisting of natural polymers and bioceramics may offer an alternative to autologous grafts for applications such as bone repair. Herein, we sought to investigate the possibility of incorporating marine coral microparticles into a collagen-based scaffold, a process which we hypothesised would enhance the mechanical properties of the scaffold as well its capacity to promote osteogenesis of human mesenchymal stromal cells. Cryomilling and sieving were utilised to achieve coral microparticles of mean diameters 14 µm and 64 µm which were separately incorporated into collagen-based slurries and freeze-dried to form porous scaffolds. X-ray diffraction and Fourier transform infrared spectroscopy determined the coral microparticles to be comprised of calcium carbonate whereas collagen/coral composite scaffolds were shown to have a crystalline calcium ethanoate structure. Crosslinked collagen/coral scaffolds demonstrated enhanced compressive properties when compared to collagen only scaffolds and also promoted more robust osteogenic differentiation of mesenchymal stromal cells, as indicated by increased expression of bone morphogenetic protein 2 at the gene level, and enhanced alkaline phosphatase activity and calcium accumulation at the protein level. Only subtle differences were observed when comparing the effect of coral microparticles of different sizes, with improved osteogenesis occurring as a result of calcium ion signalling delivered from collagen/coral composite scaffolds. These scaffolds, fabricated from entirely natural sources, therefore show promise as novel biomaterials for tissue engineering applications such as bone regeneration.

Highly versatile cell-penetrating peptide loaded scaffold for efficient and localised gene delivery to multiple cell types: From development to application in tissue engineering.

Gene therapy has recently come of age with seven viral vector-based therapies gaining regulatory approval in recent years. In tissue engineering, non-viral vectors are preferred over viral vectors, however, lower transfection efficiencies and difficulties with delivery remain major limitations hampering clinical translation. This study describes the development of a novel multi-domain cell-penetrating peptide, GET, designed to enhance cell interaction and intracellular translocation of nucleic acids; combined with a series of porous collagen-based scaffolds with proven regenerative potential for different indications. GET was capable of transfecting cell types from all three germ layers, including stem cells, with an efficiency comparable to Lipofectamine® 3000, without inducing cytotoxicity. When implanted in vivo, GET gene-activated scaffolds allowed for host cell infiltration, transfection localized to the implantation site and sustained, but transient, changes in gene expression - demonstrating both the efficacy and safety of the approach. Finally, GET carrying osteogenic (pBMP-2) and angiogenic (pVEGF) genes were incorporated into collagen-hydroxyapatite scaffolds and with a single 2 µg dose of therapeutic pDNA, induced complete repair of critical-sized bone defects within 4 weeks. GET represents an exciting development in gene therapy and by combining it with a scaffold-based delivery system offers tissue engineering solutions for a myriad of regenerative indications.