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 in vivo. We hypothesized that the unique biophysical and biochemical properties of bone mineral phase has a role in programming MSCs. Using a biomimetic bone like apatite (BBHAp) as surrogate for bone mineral phase, we studied the chondrogenic differentiation of human marrow derived MSCs and observed that the BBHAp dictates MSCs fate and strictly dictates the pathway of bone formation in vivo. Through exhaustive dissection of the signaling pathways at play, a prominent role of PTH1R in modulating the effects imposed by the BBHAp has been unraveled. These fundamental insights gained in how bone microenvironment might alter fate of MSCs has important implications for bone repair and regeneration therapies.

Summary Statement

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.

Summary

Nasal Chondrocytes are safe and feasible for tissue engineering approaches in articular cartilage repair.

Background

For bone grafting procedures, the use of autologous bone is considered the gold standard, as it is has a better healing capacity compared to other alternatives as allograft and synthetic bone substitutes. However, as there are several drawbacks related to autografting (infection, nerve- or vascular damage, chronic pain problems, abdominal herniation), there has been a targeted effort to improve the healing capacities of synthetic bone substitutes.

A major challenge to be faced in order to introduce cell-based therapies for bone repair into wide-spread surgical practice is to translate a research-scale production model into a manufacturing design that is reproducible, clinically effective, and economically viable. One possible means by which to achieve this goal is via a bioreactor system capable of controlling, automating, and streamlining all of the individual phases of the bone-tissue engineering process. In a first step to meeting this challenge, in this work we aimed at developing and validating a closed bioreactor system for

the efficient seeding of cells into 3-dimensional scaffolds and

Our patented bioreactor technology essentially consists of scaffolds arranged in a circular plate, which is moved in alternating directions by a linear drive unit through a cell suspension/culture medium, thus resulting in the perfusion of the cell suspension/culture medium directly through the pores of the scaffolds in alternate directions. The cultivation chamber is fully isolated from the external environment, with liquid/gas exchange achieved through aseptic interfaces.

Human bone marrow nucleated cells from 3 donors were perfused through porous ceramic discs (8 mm diameter, 4 mm thick), resulting in adhesion of the osteoprogenitor cell fraction in the ceramic scaffolds. Efficiency of cell seeding was consistently greater than 80%. Cell seeded constructs were further cultivated under perfusion for a total of 20 days, resulting in the expansion of the osteoprogenitor cells directly within the scaffold pores and maintenance of greater than 90% cell viability. Ectopic implantation of the cultivated constructs yielded abundant and reproducible formation of bone tissue, distributed throughout the scaffold pores.

The developed bioreactor provides a simple and efficient approach

to establish and maintain 3D cultures of cells into scaffolds under perfusion, and

Incorporating the bioreactor unit into a system for automated medium change and monitoring/control of culture parameters is likely to lead to the development of a closed system for the standardized production of autologous cell-based bone substitutes.

Tissue engineering techniques, combining autologous chondrocytes with biodegradable biomaterials, may offer significant advantages over current articular cartilage repair strategies. We present a series of experiments investigating the effect of 3D scaffold architecture and biomaterial composition on cartilage tissue formation in vitro and in vivo.

Porous polymer (PEGT/PBT) scaffolds with low (300/55/45) or high (1000/70/30) PEG molecular weight (MW) compositions were produced using novel solid free-form fabrication (3DF) techniques, allowing precise control over pore architecture, and conventional compression moulding (CM) foam techniques. Scaffolds were seeded with expanded human nasal chondrocytes, and cultured in vitro or implanted subcutaneously in vivo in nude mice for 4 weeks and cartilage tissue formation accessed.

3DF scaffolds contained highly accessible networks of large interconnecting pores (Ø525 μm) compared to CM scaffolds, containing complex networks of small interconnecting pores (Ø182 μm). 3DF scaffold architectures enhanced cell re-differentiation (GAG/DNA) and cartilaginous matrix accumulation compared to CM scaffolds, but only if 1000/70/30 compositions were used. Collagen type-II mRNA was significantly increased in 3DF architectures irrespective of scaffold composition. These effects were likely mediated by preferential protein adsorption to 1000/70/30 materials, promoting a spherical chondrocyte-like morphology, as well as efficient nutrient/waste exchange throughout interconnecting pores within 3DF architectures.

We observed synergistic effects of both composition and 3D scaffold architecture on human chondrocyte re-differentiation capacity, however, our data suggests that scaffold composition has a more significant influence than architecture alone. Such design criteria could be included in future scaffold architectures for repairing articular cartilage defects.