Notch-inducing hydrogels reveal a perivascular switch of mesenchymal stem cell fate.

The fate of mesenchymal stem cells (MSCs) in the perivascular niche, as well as factors controlling their fate, is poorly understood. Here, we study MSCs in the perivascular microenvironment of endothelial capillaries by modifying a synthetic 3D biomimetic poly(ethylene glycol) (PEG)-hydrogel system in vitro We show that MSCs together with endothelial cells form micro-capillary networks specifically in soft PEG hydrogels. Transcriptome analysis of human MSCs isolated from engineered capillaries shows a prominent switch in extracellular matrix (ECM) production. We demonstrate that the ECM phenotypic switch of MSCs can be recapitulated in the absence of endothelial cells by functionalizing PEG hydrogels with the Notch-activator Jagged1. Moreover, transient culture of MSCs in Notch-inducing microenvironments reveals the reversibility of this ECM switch. These findings provide insight into the perivascular commitment of MSCs by use of engineered niche-mimicking synthetic hydrogels.

Characterization of vasculogenic potential of human adipose-derived endothelial cells in a three-dimensional vascularized skin substitute.

PURPOSE: The need for clinically applicable skin substitutes continues to be a matter of fact. Hypothetically, a laboratory grown autologous skin analog with near normal architecture might be a suitable approach to yield both satisfactory functional and cosmetic long-term results. In this study, we explored the use of human endothelial cells derived from freshly isolated adipose stromal vascular fraction (SVF) in a three-dimensional (3D) co-culture model of vascularized bio-engineered skin substitute. METHODS: The SVF was isolated from human white adipose tissue samples and keratinocytes from human skin biopsies. The SVF, in particular endothelial cells, were characterized using flow cytometry and immuofluorescence analysis. Endothelial and mesenchymal progenitors from the SVF formed blood capillaries after seeding into a 3D collagen type I hydrogel in vitro. Subsequently, human keratinocytes were seeded on the top of those hydrogels to develop a vascularized dermo-epidermal skin substitute. RESULTS: Flow cytometric analysis of surface markers of the freshly isolated SVF showed the expression of endothelial markers (CD31, CD34, CD146), mesenchymal/stromal cell-associated markers (CD44, CD73, CD90, CD105), stem cell markers (CD49f, CD117, CD133), and additionally hematopoietic markers (CD14, CD15, CD45). Further analysis of white adipose-derived endothelial cells (watECs) revealed the co-expression of CD31, CD34, CD90, CD105, and partially CD146 on these cells. WatECs were separated from adipose-stromal cells (watASCs) using FACS sorting. WatASCs and watECs cultured separately in a 3D hydrogel for 3 weeks did not form any vascular structures. Only if co-cultured, both cell types aligned to develop a ramified vascular network in vitro with continuous endothelial lumen formation. Transplantation of those 3D-hydrogels onto immuno-incompetent rats resulted in a rapid connection of human capillaries with the host vessels and formation of functional, blood-perfused mosaic human-rat vessels within only 3-4 days. CONCLUSIONS: Adipose tissue represents an attractive cell source due to the ease of isolation and abundance of endothelial as well as mesenchymal cell lineages. Adipose-derived SVF cells exhibit the ability to form microvascular structures in vitro and support the accelerated blood perfusion in skin substitutes in vivo when transplanted.

Bioprinting of Stable Bionic Interfaces Using Piezoresistive Hydrogel Organoelectronics.

Bionic tissues offer an exciting frontier in biomedical research by integrating biological cells with artificial electronics, such as sensors. One critical hurdle is the development of artificial electronics that can mechanically harmonize with biological tissues, ensuring a robust interface for effective strain transfer and local deformation sensing. In this study, a highly tissue-integrative, soft mechanical sensor fabricated from a composite piezoresistive hydrogel. The composite not only exhibits exceptional mechanical properties, with elongation at the point of fracture reaching up to 680%, but also maintains excellent biocompatibility across multiple cell types. Furthermore, the material exhibits bioadhesive qualities, facilitating stable cell adhesion to its surface. A unique advantage of the formulation is the compatibility with 3D bioprinting, an essential technique for fabricating stable interfaces. A multimaterial sensorized 3D bionic construct is successfully bioprinted, and it is compared to structures produced via hydrogel casting. In contrast to cast constructs, the bioprinted ones display a high (87%) cell viability, preserve differentiation ability, and structural integrity of the sensor-tissue interface throughout the tissue development duration of 10 d. With easy fabrication and effective soft tissue integration, this composite holds significant promise for various biomedical applications, including implantable electronics and organ-on-a-chip technologies.

Repair of a Rat Mandibular Bone Defect by Hypertrophic Cartilage Grafts Engineered From Human Fractionated Adipose Tissue.

Background: Devitalized bone matrix (DBM) is currently the gold standard alternative to autologous bone grafting in maxillofacial surgery. However, it fully relies on its osteoconductive properties and therefore requires defects with healthy bone surrounding. Fractionated human adipose tissue, when differentiated into hypertrophic cartilage in vitro, was proven reproducibly osteogenic in vivo, by recapitulating endochondral ossification (ECO). Both types of bone substitutes were thus compared in an orthotopic, preclinical mandibular defect model in rat. Methods: Human adipose tissue samples were collected and cultured in vitro to generate disks of hypertrophic cartilage. After hypertrophic induction, eight samples from two donors were implanted into a mandible defect in rats, in parallel to Bio-Oss® DBM granules. After 12 weeks, the mandible samples were harvested and evaluated by Micro-CT and histology. Results: Micro-CT demonstrated reproducible ECO and complete restoration of the mandibular geometry with adipose-based disks, with continuous bone inside and around the defect, part of which was of human (donor) origin. In the Bio-Oss® group, instead, osteoconduction from the border of the defect was observed but no direct connection of the granules with the surrounding bone was evidenced. Adipose-based grafts generated significantly higher mineralized tissue volume (0.57 ± 0.10 vs. 0.38 ± 0.07, n = 4, p = 0.03) and newly formed bone (18.9 ± 3.4% of surface area with bone tissue vs. 3 ± 0.7%, p < 0.01) than Bio-Oss®. Conclusion: Our results provide a proof-of-concept that adipose-based hypertrophic cartilage grafts outperform clinical standard biomaterials in maxillofacial surgery.

Prefabrication of a large pedicled bone graft by engineering the germ for de novo vascularization and osteoinduction.

Large and complex bone defects represent challenging clinical scenarios, typically requiring autologous vascularized bone transplants. In order to bypass the numerous associated limitations, here we aimed at ectopically prefabricating a bone graft surrogate with vascular pedicle. A hollow cylinder of devitalized cancellous bone was used to define the space of a large bone substitute. This space was filled with devitalized pellets of engineered hypertrophic cartilage as bone-inducing material, in combination or not with stromal vascular fraction (SVF) of adipose tissue as source of osteoprogenitors and endothelial cells. Vascularization of the space was targeted through axial insertion of an arterio-venous (AV) bundle. Constructs were subcutaneously implanted in nude rats for 12 weeks and analyzed for bone formation and vascularization by histology and microtomography. Retrieved constructs were extensively vascularized in all conditions, with vessels sprouting from the AV bundle and reaching a higher density in the axially central volume. Bone tissue was formed through remodeling of hypertrophic cartilage, and quantitatively correlated with de novo vascularization. Our study demonstrates feasibility to prefabricate large, pedicled bone grafts in predefined shapes. The combination of an AV bundle with engineered hypertrophic cartilage provided a germ for the coupled processes of vascularization and bone formation. The demonstrated osteoinductivity of devitalized hypertrophic cartilage offers the opportunity of implementing the proposed regenerative surgery strategy through off-the-shelf materials.

Human fetal bone cells associated with ceramic reinforced PLA scaffolds for tissue engineering.

Fetal bone cells were shown to have an interesting potential for therapeutic use in bone tissue engineering due to their rapid growth rate and their ability to differentiate into mature osteoblasts in vitro. We describe hereafter their capability to promote bone repair in vivo when combined with porous scaffolds based on poly(l-lactic acid) (PLA) obtained by supercritical gas foaming and reinforced with 5 wt.% beta-tricalcium phosphate (TCP). Bone regeneration was assessed by radiography and histology after implantation of PLA/TCP scaffolds alone, seeded with primary fetal bone cells, or coated with demineralized bone matrix. Craniotomy critical size defects and drill defects in the femoral condyle in rats were employed. In the cranial defects, polymer degradation and cortical bone regeneration were studied up to 12 months postoperatively. Complete bone ingrowth was observed after implantation of PLA/TCP constructs seeded with human fetal bone cells. Further tests were conducted in the trabecular neighborhood of femoral condyles, where scaffolds seeded with fetal bone cells also promoted bone repair. We present here a promising approach for bone tissue engineering using human primary fetal bone cells in combination with porous PLA/TCP structures. Fetal bone cells could be selected regarding osteogenic and immune-related properties, along with their rapid growth, ease of cell banking and associated safety.

Fractionated human adipose tissue as a native biomaterial for the generation of a bone organ by endochondral ossification.

Many steps are required to generate bone through endochondral ossification with adipose mesenchymal stromal cells (ASC), from cell isolation to in vitro monolayer expansion, seeding into scaffolds, cartilaginous differentiation and in vivo remodeling. Moreover, monolayer expansion and passaging of ASC strongly decreases their differentiation potential. Here, we propose that adipose tissue itself can be used as scaffold for ASC expansion and endochondral ossification. Human liposuctions were fractionated and cultured for 3 weeks with proliferative medium in suspension. The resulting constructs, named Adiscaf, were compared to constructs generated with a previously developed, control approach, i.e. collagen sponges seeded with monolayer-expanded ASC. After 4 weeks of chondrogenic differentiation, Adiscaf contained cartilage tissue, characterized by glycosaminoglycans and collagen type II. After 2 additional weeks of hypertrophic differentiation, Adiscaf showed upregulation of hypertrophic markers at the gene expression and protein levels. After 8 weeks of in vivo implantation, Adiscaf resulted in ectopic bone tissue formation, including bone marrow elements. Adiscaf showed superior in vitro differentiation and in vivo performance as compared to the control paradigm involving isolation and monolayer expansion of ASC. This new paradigm exploits the physiological niche of adipose tissue and strongly suggests a higher functionality of cells inside adipose tissue after in vitro expansion. This study demonstrates that adult human adipose tissue used as a native construct can generate a bone organ by endochondral ossification. The concept could be exploited for the generation of osteogenic grafts for bone repair. STATEMENT OF SIGNIFICANCE: In this study we used adult human adipose tissue as scaffolding materials (called Adiscaf) to generate a bone organ by endochondral ossification. Adiscaf concept is based on the culture of adipose tissue cells inside their native microenvironment for the generation of osteogenic grafts for bone repair. This simplified approach overcomes several limitations linked to the current techniques in bone tissue engineering, such as isolation of cells and inadequate properties of the biomaterials used as scaffolds. In addition, the present paradigm proposes to exploit physiological niches in order to better maintain the functionality of cells during their in vitro expansion. This project not only has a scientific impact by evaluating the impact of native physiological niches on the functionality and chondrogenic differentiation of mesenchymal progenitors but also a clinical impact to generate osteogenic grafts and/or osteoinductive materials for bone regeneration and repair.

Facile fabrication of egg white macroporous sponges for tissue regeneration.

The availability of 3D sponges combining proper biochemical, biophysical, and biomechanical properties with enhanced capacity of in vivo engraftment and vascularization is crucial in regenerative medicine. A simple process is developed to generate macroporous scaffolds with a well-defined architecture of interconnected pores from chicken egg white (EW), a material with protein- and growth factor-binding features which has not yet been employed in regenerative medicine. The physicomechanical properties and degradation rates of the scaffold are finely tuned by using varying concentrations of the cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, without alteration of the biochemical traits. In vitro, EW scaffolds supported active metabolism, proliferation, and migration of human dermal fibroblasts, thereby generating uniform cellular constructs. In vivo, subcutaneous implantation in mice reveals negligible immune reaction and efficient cell and tissue ingrowth. Angiogenesis into EW scaffolds is enhanced as compared to standard collagen type I sponges used as reference material, likely due to significantly higher adsorption of the proangiogenic factor vascular endothelial growth factor. In summary, a material is presented derived by facile processing of a highly abundant natural product. Due to the efficient subcutaneous engraftment capacity, the sponges can find utilization for soft tissue regeneration.

Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix.

Materials based on synthetic polymers can be extensively tailored in their physical properties but often suffer from limited biological functionality. Here we tested the hypothesis that the biological performance of 3D synthetic polymer-based scaffolds can be enhanced by extracellular matrix (ECM) deposited by cells in vitro and subsequently decellularized. The hypothesis was tested in the context of bone graft substitutes, using polyesterurethane (PEU) foams and mineralized ECM laid by human mesenchymal stromal cells (hMSC). A perfusion-based bioreactor system was critically employed to uniformly seed and culture hMSC in the scaffolds and to efficiently decellularize (94% DNA reduction) the resulting ECM while preserving its main organic and inorganic components. As compared to plain PEU, the decellularized ECM-polymer hybrids supported the osteoblastic differentiation of newly seeded hMSC by up-regulating the mRNA expression of typical osteoblastic genes (6-fold higher bone sialoprotein; 4-fold higher osteocalcin and osteopontin) and increasing calcium deposition (6-fold higher), approaching the performance of ceramic-based materials. After ectopic implantation in nude mice, the decellularized hybrids induced the formation of a mineralized matrix positively immunostained for bone sialoprotein and resembling an immature osteoid tissue. Our findings consolidate the perspective of bioreactor-based production of ECM-decorated polymeric scaffolds as off-the-shelf materials combining tunable physical properties with the physiological presentation of instructive biological signals.