Matrix-bound vesicles (MBVs) are embedded within osteoid and function as the site of initial mineral formation. However, they remain insufficiently characterised in terms of biogenesis, composition and function while their relationship with secreted culture medium EVs (sEVs) such as exosomes remains debated. We aimed to define the biogenesis and pro-mineralisation capacity of MBVs and sEVs to understand their potential in regenerative orthopaedics. sEVs and MBVs isolated from conditioned medium (differential ultracentrifugation) and ECM (collagenase digestion and differential ultracentrifugation) of mineralising MC3T3 pre-osteoblast and human bone marrow MSC cultures were characterised by nanoparticle tracking analysis, western blotting, nano-flow cytometry, super resolution microscopy (ONI) and TEM. Immunoprecipitated populations positive for alkaline phosphatase (ALP), a putative marker of mineralisation capacity, were also characterised. Collagen binding efficiency was evaluated using MemGlow staining. Results reported were comparative across both cell lines. Western blots indicated MBV fractions were positive for markers of endosomal biogenesis (CD9, CD81, ALIX, TSG101) and pro-mineralising proteins (ALP, Pit1, Annexin II, Annexin V), with Annexin V and CD9 present in immunoprecipitated ALP-positive fractions. MBVs were significantly larger than sEVs (p<0.05) and contained a higher amount of ALP (p<0.05) with a significant increase from day 7 to day 14 of cellular mineralisation (p<0.05). This mirrored the pattern of electron-dense vesicles seen via TEM. Super resolution single vesicle analysis revealed for the first-time co-expression of ALP with markers of endosomal biogenesis (CD9, CD63, CD81, ALIX) and Annexin II in both vesicle types, with higher co-expression percentage in MBVs than sEVs. MBVs also exhibited preferential collagen binding. Advanced imaging methods demonstrated that contrary to opinions in the field, MBVs appear to possess exosomal markers and may arise via endosomal biogenesis. However, it was evident that a higher proportion of MBVs possessed machinery to induce mineralisation and were enriched in mineral-dense material.
Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, we report the manufacturing of engineered and devitalized human hypertrophic cartilage (HyC) as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification. Our strategy relies on a customized human mesenchymal line expressing Bone Morphogenetic Protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization and storage, the resulting material exhibited unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy were demonstrated by bioreactor-based production and subsequent orthotopic assessment. Our findings exemplify the broader paradigm of customized ECMs, engineered to activate specific regenerative processes by programming human cell lines as biological factory units.
Bone formation proceeds through two distinct processes. One involves the deposition of bone by osteoblasts (intramembranous ossification) and another through the remodeling of an intermediate cartilaginous matrix formed by chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs) aggregates – a process called endochondral ossification (EO). EO is responsible for formation of long bones during development and most prevalent during facture repair upon callus formation. In adult bone injuries MSCs from periosteum form bone via EO whereas MSCs from bone marrow are primarily differentiate to osteoblast
This study reports that hMSC can be manipulated in order to engineer a bone organ, characterised by mature osseous and vascular components and capable to recruit, host and maintain functional HSCs. Bone tissue engineering strategies are typically based on methods involving adult human Mesenchymal Stromal Cells (hMSC) in a process resembling intramembranous ossification. However, most bones develop and repair through endochondral ossification. In addition, endochondral ossification presents several advantages for regenerative purposes such as osteogenic activity, capability to drive formation of the Hematopoietic Stem Cell (HSC) niche, resistance to hypoxia, intrinsic vasculogenic potential and, consequently, efficiency of engraftment. In this study, we aimed at developing an endochondral bone organ model characterised by functional osseous and hematopoietic compartments by using hMSC.Summary Statement
Introduction
Nasal Chondrocytes are safe and feasible for tissue engineering approaches in articular cartilage repair. As compared to articular chondrocytes (AC), nasal septum chondrocytes (NC) proliferate faster and have a higher and more reproducible capacity to generate hyaline-like cartilaginous tissues. Moreover, the use of NC would allow reducing the morbidity associated with the harvesting of cartilage biopsy from the patient. The objective of the present study was to demonstrate safety and feasibility in the use of tissue engineered cartilage graft based on autologous nasal chondrocytes for the repair of articular defect in goats.Summary
Introduction
