Biology subjects

Kronemberger, G. S.

Publications and source records attributed to Kronemberger, G. S..

3 recordsLinked to original sources

bioRxiv2026

Integrating vascular and hypertrophic cartilage microtissues to fabricatescaled-up grafts for endochondral bone tissue engineering

ABSTRACTThe repair of large bone defects remains a major clinical challenge, in part due to inadequate vascularization and poor integration of graft materials. Tissue engineering strategies that recapitulate the developmental process of endochondral ossification, whereby a cartilage template remodels into bone, have shown significant potential in pre-clinical models of large bone defect healing. However, successfully scaling these approaches to clinically relevant sizes will require the development of strategies to support the rapid vascularization of the graft following implantation in vivo. Here, mechanically reinforced templates were first fabricated by integrating hypertrophic cartilage microtissues derived from human mesenchymal stem/stromal cells (MSCs) within an osteoconductive 3D-printed polycaprolactone (PCL) framework coated with nano-hydroxyapatite (nanoHA). In vitro the cartilage microtissues fused and generated an extracellular matrix rich in sulphated glycosaminoglycans and collagen. To prevascularize these constructs, vascular microtissues derived from a co-culture of endothelial cells and MSCs were incorporated into a central channel within the construct, which generated a microvascular network within the graft in vitro. Following subcutaneous implantation, hypertrophic cartilage templates with ( vascular-channel group) and without ( empty-channel group) this central vascularized channel supported endochondral bone formation. Quantitative microCT and histological analyses revealed significantly greater remaining bone in the empty-channel group, whereas the vascular-channel group supported enhanced vascularization and remodeling of the graft in vivo. These findings support the continued development and testing of a modular biofabrication strategy that combine self-organizing hypertrophic cartilage and vascular microtissues with osteoconductive 3D-printed architectures to generate scalable, prevascularised hypertrophic cartilage templates for endochondral bone repair. Key-words: spheroids, microtissues, hypertrophic cartilage, vascularization, endochondral ossification, bone tissue engineering.

bioengineering↗

bioRxiv2025

Bioassembly of region-specific fibrocartilage microtissues to engineer zonally defined meniscal grafts

Meniscal injuries are common orthopaedic problems which can impair knee function and lead to the development of osteoarthritis. While recent advances in tissue engineering have enabled the fabrication of meniscus-like grafts, these constructs do not fully replicate the zonal structure and composition of the native meniscus. In this study, we used fibrocartilage microtissues generated from meniscus progenitor cells (MPCs) as biological building blocks to biofabricate zonally defined meniscal grafts. MPCs isolated from the inner (iMPC) and outer (oMPC) regions of caprine menisci were used to engineer region-specific meniscal microtissues in a medium-high throughput manner. Both iMPC and oMPC derived microtissues were rich in glycosaminoglycans (GAGs) and collagen, with iMPC microtissues staining more intensely for type II collagen. These microtissues were assembled into two different physically confining moulds (cylindrical and ring-shaped), where they rapidly fused and generated a fibrocartilaginous graft over six weeks of culture. Both iMPC and oMPC assembled microtissues were rich in sGAG and type I collagen, however only the iMPC assembled microtissues stained strongly for type II collagen. We then explored the impact of the catabolic enzyme chondroitinase-ABC (cABC) on the composition and structural organization of the meniscal grafts. This temporal enzymatic treatment increased collagen fiber thickness without altering tissue phenotype. Finally, iMPC and oMPC microtissues were spatially assembled to biofabricate a scaled-up and zonally defined meniscal graft. This graft was phenotypically similar to the native meniscus, with all regions rich in collagen type I and an inner core rich in collagen type II and sGAG. These findings support the use of MPC derived microtissues as biological building blocks for the engineering of zonally defined meniscal grafts. Table of ContentO_LIZonally defined meniscus grafts were biofabricated using inner and outer meniscus progenitor cells microtissues. C_LIO_LIRegion specific microtissues showed phenotypes similar to the native meniscus and successfully fused into cohesive constructs rich in sGAG and collagen. C_LIO_LIcABC treatment modulated collagen fiber formation and organization in assembled grafts. C_LIO_LIThe assembled grafts maintained shape fidelity and may offer a promising strategy for meniscal repair. C_LI O_FIG O_LINKSMALLFIG WIDTH=132 HEIGHT=200 SRC="FIGDIR/small/650568v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1354a5forg.highwire.dtl.DTLVardef@1a5acd3org.highwire.dtl.DTLVardef@1f558aborg.highwire.dtl.DTLVardef@90666e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Biofabrication of zonally defined meniscal grafts using meniscus progenitor cell (MPC)-derived microtissues. (A) Meniscus progenitor cells (MPCs) derived from the inner (iMPC), and outer (oMPC) regions of the menisci were aggregated into microtissues by cellular self-assembly process using a high-throughput agarose microwell system developed in-house. (B) The iMPC and oMPC microtissues were then separately fused in cylindrical and ring-shaped moulds to form region-specific assembled microtissues. (C) To enhance structural organization, chondroitinase ABC (cABC) enzymatic treatment was applied to the ring-shaped assembled microtissues. (D) Finally, the matured iMPC and oMPC assembled microtissues were assembled to create a scaled-up and zonally defined meniscal graft. C_FIG

bioengineering↗

bioRxiv2022

Engineering High-Quality Cartilage Microtissues using Hydrocortisone Functionalised Microwells

Engineering clinically-relevant musculoskeletal tissues at a human scale is a considerable challenge. Developmentally-inspired scaffold-free approaches for engineering cartilage tissues have shown great promise in recent years, enabling the generation of highly biomimetic tissues. Despite the relative success of these approaches, the absence of a supporting scaffold or hydrogel creates challenges in the development of large scale tissues. Combining numerous scaled-down tissue units (herein termed microtissues) into a larger macrotissue represents a promising strategy to address this challenge. The overall success of such approaches, however, relies on the development of strategies to support the robust and consistent chondrogenic differentiation of clinically relevant cell sources such as mesenchymal stem/stromal cells (MSCs) within microwell arrays to biofabricate numerous microtissues rich in cartilage-specific extracellular matrix components. In this paper, we first describe a simple method to manufacture cartilage microtissues at various scales using novel microwell array stamps. This system allows the rapid and reliable generation of cartilage microtissues, and can be used as a platform to study microtissue phenotype and development. Based on the unexpected discovery that Endothelial Growth Medium (EGM) enhanced MSC aggregation and chondrogenic capacity within the microwell arrays, this work also sought to identify soluble factors within the media capable of supporting robust differentiation using heterogeneous MSC populations. Hydrocortisone was found to be the key factor within EGM that enhanced the chondrogenic capacity of MSCs within these microwell arrays. This strategy represents a promising means of generating large numbers of high-quality, scaffold-free cartilage microtissues for diverse biofabrication applications.

bioengineering↗