Mesenchymal stem cell-mediated endochondral ossification utilising micropellets and brief chondrogenic priming

Abstract

With limited autologous and donor bone graft availability, there is an increasing need for alternative graft substitutes. We have previously shown that chondrogenically priming mesenchymal stem cell (MSC) pellets for 28 d in vitro will reproducibly result in endochondral bone formation after in vivo implantation. However, pellet priming time for clinical applications is quite extensive. A micropellet (μpellet)-fibrin construct was developed and coupled, with a shorter priming period, determined by an in vitro time course experiment. In vitro data showed expression of chondrogenic genes and matrix production after 7 d of chondrogenic priming, indicating that briefer priming could possibly be used to induce bone formation in vivo. 7 and 28 d primed pellet, pellet-fibrin and μpellet-fibrin constructs were cultured for in vitro analysis and implanted subcutaneously for 8 weeks into nude mice. μpellet-fibrin constructs, cultured in vitro for 7 or 28 d, produced comparable bone to standard pellets in vivo. MSC-mediated bone formation was achieved following only 7 d of in vitro priming. Bone formation in vivo appeared to be influenced by overall matrix production pre-implantation. Given this short priming time and the injectable nature of the μpellet-fibrin constructs, this approach might be further developed as an injectable bone substitute, leading to a minimally-invasive treatment option, which would allow for tailored filling of bone defects.

Single-cell characterization and metabolic profiling of in vitro cultured human skeletal progenitors with enhanced in vivo bone forming capacity

“…This is of notable interest for pleiotropic growth factors such as BMP ligands, for which the safety has been debated in recent years due to the implantation of supraphysiological doses . The potency of solely in vitro priming on the other hand has shown limited success due to insufficient tissue formation in vivo , likely due to a heterogeneous and noncommitted progenitor population . In attempts to overcome this, prolonged differentiation time increases the maturity thus potentially also in vivo tissue formation, but challenges integration at the defect site upon implantation .…”

Understanding tissue-engineered endochondral ossification; towards improved bone formation

“…Roselló Llabrés et al, 2014 www.ecmjournal.org C Knuth et al Tissue-engineered endochondral ossification bone formation following implantation. Knuth et al (2017) reported how stronger chondrogenic induction can influence in vivo bone formation; however, they hypothesised that a more GAGrich matrix delays bone marrow formation due to delayed remodelling. Perhaps this indicates that parameters, using ECM components produced by chondrogenically-differentiated MSCs, can be set to assess bone formation; nevertheless, performing this without destroying the pellet would be difficult.…”

“…The coordination of these events with cell/ vascular recruitment ultimately controls effective www.ecmjournal.org bone formation in EO. This can be recapitulated in TERM by differentiating MSCs chondrogenically and implanting them subcutaneously either as pellets or seeded in scaffolds (Knuth et al, 2017;Scotti et al, 2010;Tonnarelli et al, 2014;van der Stok et al, 2014). This seems to mirror developmental EO and shows excellent integration within the host tissue (Chan et al, 2009).…”

The impact of immune response on endochondral bone regeneration

“…The feasibility of recapitulating the above described natural healing process for regenerative purposes has been widely explored in the last decades. 4 , 7 , 11 – 15 , 43 , 44 Several studies demonstrated that in vitro engineered cartilage templates obtained from MSCs alone, 13 , 19 , 44 or in combination with different biomaterials, 14 , 15 , 43 , 45 could be successfully converted into new bone tissue upon implantation, both ectopically 12 , 19 and orthotopically. 13 , 14 , 43 , 45 However, so far no consensus has been reached regarding the optimal length of the period for chondrogenic differentiation prior to implantation.…”

“… 13 , 14 , 43 , 45 However, so far no consensus has been reached regarding the optimal length of the period for chondrogenic differentiation prior to implantation. 4 , 46 It could span from as little as 1 week 44 to 7 weeks. 19 Also, no agreement exists regarding the optimal differentiation status (chondrogenic or hypertrophic chondrogenic) before implantation.…”