Stem Cell and Regenerative Medicine
We have been optimizing the scaffolds, modifying chondroinducing medium, as well as modulating the relevant signaling pathways to induce chondrocyte MSCs or iPSCs creating hyaline cartilage for the repair of chondral injury.
Condensation-Driven Chondrogenesis of Human Mesenchymal Stem Cells within Their Own Extracellular Matrix
Human mesenchymal stem cells (MSCs) are cultured under confluent conditions for the deposition of the ECM (mECM). Subsequently, the MSC‐impregnated mECM composite is briefly treated with trypsin, allowing the MSCs to adopt a round morphology without being detached from their own mECM. Interestingly, after trypsin removal, the treated MSCs undergo mesenchymal condensation, followed by robust chondrogenic differentiation.
We have been using ECM derived from mesenchymal stem cells since 2012. A review article was published in 2020 to summarize the application of MSC-ECM in tissue engineering. Other relevant work was published in 2018 and 2019.
Infiltration and In-Tissue Polymerization of Hydrogel for Effective Fixation of Implants into Cartilage
Effective and biocompatible fixation of implants into cartilage defects has yet to be successfully achieved. [Poly-d,l-lactic acid/polyethyleneglycol/poly-d,l-lactic acid] (PDLLA-PEG) is a chondrosupportive scaffold that is photocross-linked using the visible-light photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Interestingly, LAP and its monomer DLLA-EG are able to infiltrate the cartilage and form hydrogels upon the detection of light. After the infiltration of LAP and DLLA-EG into the implant and host cartilage, an interconnected and continuous hydrogel structure is formed which fixes the implant within the host cartilage. A mechanical test shows that the infiltrated group displays a significantly higher push-out force than the group that has not been infiltrated (the traditional fibrin fixation group). Surprisingly, the in-cartilage hydrogel also reduces the release of sulfated glycosaminoglycan from cartilage explants. This new strategy thus represents a biocompatible and efficient method to fix implants into host tissues.
Photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect
Hybrid scaffolds consisting of visible light-activatable methacrylated gelatin (mGL) and methacrylated hyaluronic acid (mHA) hydrogel at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 106 cells/ml and maintained in a chondrogenesis-promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full-thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three-dimensional cell-biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.
We have been using visible light-based, photocrosslinkable biomaterials since 2013. Injectable gelatin hydrogel with in situ visible-light-activated gelation capability was developed in 2014. Other relevant work was published in 2019, 2020.
Engineering hyaline cartilage from mesenchymal stem cells with low hypertrophy potential
Mesenchymal stem cells (MSCs) represent a promising cell source to regenerate articular cartilage, but current chondroinduction protocols, commonly using transforming growth factor-β (TGFβ), lead to concomitant chondrocytic hypertrophy with ossification risk. Here, we showed that a 14-day culture of MSC-laden hyaluronic acid hydrogel in the presence of TGFβ, followed by 7 days culture in TGFβ-free medium, with the supplement of Wnt/β-catenin inhibitor XAV939 from day 10-21, resulted in significantly reduced hypertrophy phenotype. The stability of the hyaline phenotype of the MSC-derived cartilage, generated with a standard protocol (Control) or the optimized (Optimized) method developed in this study, was further examined through intramuscular implantation in nude mice. After 4 weeks, constructs from the Control group showed obvious mineralization; in contrast, the Optimized group displayed no signs of mineralization, and maintained cartilaginous histology. Further analysis showed that TGFβ treatment time affected p38 expression, while exposure to XAV939 significantly inhibited P-Smad 1/5 level, which together resulted in decreased level of Runx2.
Application of visible light-based projection stereolithography for live cell-scaffold fabrication with designed architecture
One-step scaffold fabrication with live cell incorporation is a highly desirable technology for tissue engineering and regeneration. Projection stereolithography (PSL) represents a promising method owing to its fine resolution, high fabrication speed and computer-aided design (CAD) capabilities. However, the majority of current protocols utilize water-insoluble photoinitiators that are incompatible with live cell-fabrication, and ultraviolet (UV) light that is damaging to the cellular DNA. We report here the development of a visible light-based PSL system (VL-PSL), using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as the initiator and polyethylene glycol diacrylate (PEGDA) as the monomer, to produce hydrogel scaffolds with specific shapes and internal architectures. Furthermore, live human adipose-derived stem cells (hADSCs) were suspended in PEGDA/LAP solution during the PSL process, and were successfully incorporated within the fabricated hydrogel scaffolds. hADSCs in PEG scaffolds showed high viability (>90%) for up to 7 days after fabrication as revealed by Live/Dead staining. Scaffolds with porous internal architecture retained higher cell viability and activity than solid scaffolds, likely due to increased oxygen and nutrients exchange into the interior of the scaffolds.
Rejuvenation of Aged Human Articular Chondrocytes for High-quality Cartilage Regeneration
Chondrocytes isolated from aged donors displayed reduced proliferation potential and impaired capacity in generating hyaline cartilage, compared to cells isolated from young donors, indicated by increased hypertrophy and cellular senescence. To test our hypothesis, the “old” chondrocytes were seeded onto an agarose-based substratum, where they maintained a round morphology. After the 3-day suspension culture, aged chondrocytes displayed enhanced replicative capacity, compared to those grown adherent to tissue culture plastic. Moreover, chondrocytes subjected to suspension culture formed new cartilage in vitro with higher quality and quantity, with enhanced cartilage matrix deposition, concomitant with lower levels of hypertrophy and cellular senescence markers. Mechanistic analysis suggested the involvement of the RhoA and ERK1/2 signaling pathways in the "rejuvenation" process. In summary, our study presents a robust and straightforward method to enhance the function of aged human chondrocytes, which can be conveniently used to generate a large number of high-quality chondrocytes for ACI application in the elderly.