Biology subjects

Alt, D. S.

Publications and source records attributed to Alt, D. S..

4 recordsLinked to original sources

bioRxiv2025

Three-dimensional tissue platform co-laid with native collagen fibers and cells for phenotypic screening of stem cell interactions

Phenotypic screening of cell-cell and cell-matrix interactions is critical yet challenging for drug discovery and disease modeling. In this study, a scalable 3D tissue platform was developed by co-laying extracted natural insoluble collagen fibers, mesenchymal stem cells, endothelial cells, and neural progenitor cells for phenotypic screening. Cell growth and interactions were enhanced in the co-laid platform, evident through increased cell proliferation, viability, and vascularization. Dense vascular networks rapidly formed through cell-cell and cell-matrix interactions without adding a traditionally needed growth factor set. Both in vitro and implantation studies confirmed that these blood vessels were of human origin. To evaluate the phenotypic screening of cell-cell and cell-matrix interactions, we propose a phenotype screening prototype for stem cell interactions that utilized multivariate analysis encompassing both cell-cell and cell-matrix interactions and demonstrated its effectiveness to screen vasculature formation and autism spectrum disorder (ASD) models. Using the prototype, we confirmed that collagen crosslinking, ROCK, WNT, and YAP pathways impact vasculogenesis. In addition, ASD donor-derived neural progenitor cells can be distinguished from non-ASD control donor-derived neural progenitor cells.

bioengineering↗

bioRxiv2024

In situ cell condensation-based cartilage tissue engineering via immediately implantable high-density stem cell core and rapidly degradable shell microgels

Formation of chondromimetic human mesenchymal stem cells (hMSCs) condensations typically required in vitro culture in defined environments. In addition, extended in vitro culture in differentiation media over several weeks is usually necessary prior to implantation, which is costly, time consuming and delays clinical treatment. Here, this study reports on immediately implantable core/shell microgels with a high-density hMSC-laden core and rapidly degradable hydrogel shell. The hMSCs in the core formed cell condensates within 12 hours and the oxidized and methacrylated alginate (OMA) hydrogel shells were completely degraded within 3 days, enabling spontaneous and precipitous fusion of adjacent condensed aggregates. By delivering transforming growth factor-{beta}1 (TGF-{beta}1) within the core, the fused condensates were chondrogenically differentiated and formed cartilage microtissues. Importantly, these hMSC-laden core/shell microgels, fabricated without any in vitro culture, were subcutaneously implanted into mice and shown to form cartilage tissue via cellular condensations in the core after 3 weeks. This innovative approach to form cell condensations in situ without in vitro culture that can fuse together with each other and with host tissue and be matured into new tissue with incorporated bioactive signals, allows for immediate implantation and may be a platform strategy for cartilage regeneration and other tissue engineering applications.

bioengineering↗

bioRxiv2019

Scaffold-free human mesenchymal stem cell construct geometry regulates long bone regeneration

Scaffold-based bone tissue engineering approaches frequently induce repair processes dissimilar to normal developmental programs. In contrast, biomimetic strategies aim to recapitulate aspects of development through cellular self-organization, morphogenetic pathway activation, and mechanical cues. This may improve regenerative outcome in large long bone defects that cannot heal on their own; however, no study to date has investigated the role of scaffold-free construct geometry, in this case tubes mimicking long bone diaphyses, on bone regeneration. We hypothesized that microparticle-mediated in situ presentation of transforming growth factor-{beta}1 (TGF-{beta}1) and bone morphogenetic protein-2 (BMP-2) to engineered human mesenchymal stem cell (hMSC) tubes induces the endochondral cascade, and that TGF-{beta}1 + BMP-2-presenting hMSC tubes facilitate enhanced endochondral healing of critical-sized femoral segmental defects under delayed in vivo mechanical loading conditions compared to loosely-packed hMSC sheets. Here, localized morphogen presentation imparted early chondrogenic lineage priming, and stimulated robust endochondral differentiation of hMSC tubes in vitro. In an ectopic environment, hMSC tubes formed a cartilage template that was actively remodeled into trabecular bone through endochondral ossification without lengthy predifferentiation. Similarly, hMSC tubes stimulated in vivo cartilage and bone formation and more robust healing in femoral defects compared to hMSC sheets. New bone was formed through endochondral ossification in both groups; however, only hMSC tubes induced regenerate tissue partially resembling normal growth plate architecture. Together, this study demonstrates the interaction between mesenchymal cell condensation geometry, bioavailability of multiple morphogens, and defined in vivo mechanical environment to recapitulate developmental programs for biomimetic bone tissue engineering.\n\nSignificance StatementEngineered bone constructs must be capable of withstanding and adapting to harsh conditions in a defect site upon implantation, and can be designed to facilitate repair processes that resemble normal developmental programs. Self-assembled tubular human mesenchymal stem cell constructs were engineered to resemble the geometry of long bone diaphyses. By mimicking the cellular, biochemical, and mechanical environment of the endochondral ossification process during embryonic development, successful healing of large femoral segmental defects upon implantation was achieved and the extent was construct geometry dependent. Importantly, results were obtained without a supporting scaffold or lengthy predifferentiation of the tubular constructs. This indicates that adult stem/progenitor cells retain features of embryonic mesenchyme, and supports the concept of developmental engineering for bone regeneration approaches.

bioengineering↗

bioRxiv2019

Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-{beta}1 (TGF-{beta}1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-{beta}1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-{beta}1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-{beta}1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering. One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

bioengineering↗