Futrega, K. | Search bioRxiv

bioRxiv2020

Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo

BackgroundBone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model. The objective of this study was to characterize ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep. MethodsoBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. Expanded oBMSC were positive for CD44 and CD146 and negative for CD45. ResultsThe common adipogenic induction medium ingredient, 3-Isobutyl-1-methylxanthine (IBMX) was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not. ConclusionThe sensitivity of oBMSC to IBMX highlights species-species differences between oBMSC and hBMSC. Micro-pellets manufactured from oBMSC were not effective in repairing osteochondral defects, while oACh micro-pellets enabled modest repair. While oBMSC can be driven to form cartilage-like tissue in vitro, their effective use in cartilage repair will require mitigation of hypertrophy.

bioengineering↗

bioRxiv2019

Micro-pellet culture reveals that bone marrow mesenchymal stromal cell (BMSC) chondrogenic induction is triggered by a single day of TGF-β1 exposure

Despite immense promise, engineering of stable cartilage tissue from bone marrow-derived stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") remains elusive. Relative cartilage-like matrix deposition is commonly used to guide BMSC chondrogenic optimisation efforts. However, matrix deposition is heterogeneous in most models, and notably, it lags behind cell fate decisions. We reason that the lag time between cell fate decision and matrix accumulation, coupled with matrix heterogeneity, has obscured basic BMSC biological characteristics, such as differentiation kinetics. Here, we utilize a customized microwell platform to assemble hundreds of small-diameter BMSC micro-pellets and characterized chondrogenic differentiation kinetics in response to the canonical signaling molecule, transforming growth factor-{beta}1 (TGF-{beta}1). Micro-pellets provide a homogeneous readout, and our experimental design accounts for the significant time delay between growth factor signal and deposition of cartilage-like matrix. While 14-to-21-day induction protocols are routine, BMSC micro-pellet cultures reveal that a single day of TGF-{beta}1 exposure was sufficient to trigger chondrogenic differentiation cascades resulting in outcomes similar to micro-pellets exposed to TGF-{beta}1 for 21 days. RNA-sequencing analysis demonstrated that one day of TGF-{beta}1 exposure was also sufficient to induce hypertrophic cascades in BMSC, not observed in articular chondrocytes. Refocusing chondrogenic induction optimisation efforts from weeks to the first hours or days of culture, using homogeneous model systems, may benefit efforts to build stable cartilage formed by BMSCs. SignificanceThe macro-pellet model, and assumptions generated using it, have permeated BMSC-based cartilage tissue engineering strategies since the 1990s. Using a micro-pellet model, we show that BMSC chondrogenic kinetics are significantly more rapid than historical macro-pellets data suggests, and that BMSC chondrogenic and hypertrophic commitment is instructed by a single day of TGF-{beta}1 exposure. This highly relevant study demonstrates that: (1) macro-pellets, which are large heterogeneous tissue models confound the differentiation kinetics visible in micro-pellet models; (2) induction strategies should focus on the first hours or days of culture; (3) even a single day of TGF-{beta}1 exposure drives BMSC to form hypertrophic tissue in vivo, requiring early intervention to prevent hypertrophy; and (4) articular chondrocytes and BMSCs respond distinctly to TGF-{beta}1.

cell biology↗