Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR + cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.

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.

Mesenchymal stem cells/marrow stromal cells (MSCs) are attractive for applications ranging from research and development to use in clinical therapeutics. However, the most commonly studied MSCs, adult bone marrow MSCs (A-MSCs), are limited by significant donor variation resulting in inconsistent expansion rates and multilineage differentiation capabilities. We have recently obtained permission to isolate pediatric MSCs (P-MSCs) from surplus iliac crest bone chips. Here, we developed a simple and easily replicable isolation protocol yielding P-MSCs, which adhere to MSC defining guidelines. After confirming immunophenotypic marker expression, we compared expansion rates, senescence, morphology, and trilineage differentiation of P-MSCs to A-MSCs for multiple donors. We found P-MSCs have faster in vitro replication, consistently show significantly lower senescence, and are capable of more reproducible multilineage differentiation than A-MSCs. We, therefore, believe P-MSCs are a promising candidate for use in research applications and potentially as part of an allogeneic therapeutic treatment.

In tissue engineering, endochondral ossification (EO) is often replicated by chondrogenically differentiating mesenchymal stromal cells (MSCs) in vitro and achieving bone formation through in vivo implantation. The resulting marrow-containing bone constructs are promising as a treatment for bone defects. However, limited bone formation capacity has prevented them from reaching their full potential. This is further complicated since it is not fully understood how this bone formation is achieved. Acellular grafts derived from chondrogenically differentiated MSCs can initiate bone formation; however, which component within these decellularised matrices contribute to bone formation has yet to be determined. Collagen type X (COLX), a hypertrophy-associated collagen found within these constructs, is involved in matrix organisation, calcium binding and matrix vesicle compartmentalisation. However, the importance of COLX during tissue-engineered chondrogenesis and subsequent bone formation is unknown. The present study investigated the importance of COLX by shRNA-mediated gene silencing in primary MSCs. A significant knock-down of COLX disrupted the production of extracellular matrix key components and the secretion profile of chondrogenically differentiated MSCs. Following in vivo implantation, disrupted bone formation in knock-down constructs was observed. The importance of COLX was confirmed during both chondrogenic differentiation and subsequent EO in this tissue engineering setting.

Satisfactory osseous tissue integration of the soft tissue graft with bone is the mainstay of healing following surgical reconstruction of the anterior cruciate ligament (ACL). However, tissue remodelling is slow and significantly impacts on quality of life by delaying return to work and sport and accelerating the onset of degenerative diseases such as osteoarthritis. Delivery of multipotent human mesenchymal stem cells (hMSCs) at surgery could enhance osseous tissue integration. We aim to use hMSCs derived from haemarthrosis fluid (HF) (the intra-articular bleed accrued post-trauma) which is aspirated and discarded as clinical waste. With the aim of improving our bioprocessing methodologies for clinical translation we have investigated the effect of low oxygen tension on the derivation and osteogenic potential of this novel HF-hMSC population. Mononuclear cells were isolated from HF aspirated samples and divided for derivation and culture under normal or low oxygen tension. HF-hMSCs were derived from 100 % of cultures under low oxygen tension compared to 71 % for normal oxygen tension; this was coupled with increased CFU-Fs. We investigated the osteogenic potential and cellular health of HF-hMSC populations following ex vivo expansion. HF-hMSC populations showed enhanced matrix mineralisation and cellular health when differentiated under low oxygen tension. This positive effect of low oxygen on osteogenesis and cellular health was reduced with prolonged culture. These data demonstrate that derivation and culture of HF-hMSC populations under low oxygen tension will enable the translation of a cellular therapy for the treatment of broad patient numbers with optimal osteogenic potency and cellular vitality.

Tissue engineering strategies can be applied to enhancing osseous integration of soft tissue grafts during ligament reconstruction. Ligament rupture results in a hemarthrosis, an acute intra-articular bleed rich in osteogenic human mesenchymal stem cells (hMSCs). With the aim of identifying an appropriate biomaterial with which to combine hemarthrosis fluid-derived hMSCs (HF-hMSCs) for therapeutic application, this work has investigated the biocompatibility of microparticles manufactured from two forms of poly(D,L-lactic-co-glycolic acid) (PLGA), one synthesized with equal monomeric ratios of lactic acid to glycolic acid (PLGA 50:50) and the other with a higher proportion of lactic acid (PLGA 85:15) which confers a longer biodegradation time. The surfaces of both types of microparticles were functionalized by plasma polymerization with allylamine to increase hydrophilicity and promote cell attachment. HF-hMSCs attached to and spread along the surface of both forms of PLGA microparticle. The osteogenic response of HF-hMSCs was enhanced when cultured with PLGA compared with control cultures differentiated on tissue culture plastic and this was independent of the type of polymer used. We have demonstrated that surface engineered PLGA microparticles are an appropriate biomaterial for combining with HF-hMSCs and the selection of PLGA is relevant only when considering the biodegradation time for each biomedical application.

No abstract

Background: Treatment of large bone defects due to trauma, tumor resection, or congenital abnormalities is challenging. Bone tissue engineering using mesenchymal stem cells (MSCs) represents a promising treatment option. However, the quantity and quality of engineered bone tissue are not sufficient to fill large bone defects. The aim of this study was to determine if the addition of enamel matrix derivative (EMD) improves in vitro chondrogenic priming of MSCs to ultimately improve in vivo MSC mediated endochondral bone formation.Methods: MSCs were chondrogenically differentiated in 2.0 × 105 cell pellets in medium supplemented with TGFβ3 in the absence or presence of 1, 10, or 100 μg/mL EMD. Samples were analyzed for gene expression of RUNX2, Col II, Col X, and Sox9. Protein and glycoaminoglycan (GAG) production were also investigated via DMB assays, histology, and immunohistochemistry. Osteogenic and adipogenic differentiation capacity were also assessed.Results: The addition of EMD did not negatively affect chondrogenic differentiation of adult human MSCs. EMD did not appear to alter GAG production or expression of chondrogenic genes. Osteogenic and adipogenic differentiation were also unaffected though a trend toward decreased adipogenic gene expression was observed.Conclusion: EMD does not affect chondrogenic differentiation of adult human MSCs. As such the use of EMD in combination with chondrogenically primed MSCs for periodontal bone tissue repair is unlikely to have negative effects on MSC differentiation.