Cocultivation of Mesenchymal Stem Cells and Endothelial Progenitor Cells Reveals Antiapoptotic and Proangiogenic Effects.

Integrating bioartificial tissues into the host vasculature is a prerequisite for tissue engineering applications. Endothelial progenitor cells (EPCs) display a high angiogenic potential and a low donor-site morbidity, making them ideal for tissue engineering applications. In our study we used a murine EPC cell line (T17b) and rat mesenchymal stem cells (MSCs) for cocultivation experiments. MSCs were cocultured with increasing T17b EPC amounts. Furthermore, MSCs in monoculture were treated with conditioned medium (CM) from T17b EPCs and T17b EPCs were treated with CM from MSCs. Proliferation and apoptosis were quantified with a bromodeoxyuridine ELISA and a DNA fragmentation ELISA, respectively. Osteogenic differentiation was detected with an alkaline phosphatase assay and bone morphogenetic protein-2 ELISA. The production of proangiogenic molecules was measured with a matrix metalloproteinase-3 and vascular endothelial growth factor ELISA as well as nitric oxide assay. We could show that T17b EPCs stimulated MSC proliferation but not vice versa. On the other hand, MSCs promoted the cell survival of EPCs. The growth-inducing and antiapoptotic effects were dependent on heterotypic cell contacts and paracrine mediators. Moreover, proangiogenic growth factors were found in the coculture. Collectively, our results indicate that the coapplication of MSCs and T17b EPCs provides new perspectives for tissue engineering applications.

Free Transplantation of a Tissue Engineered Bone Graft into an Irradiated, Critical-Size Femoral Defect in Rats.

Healing of large bone defects remains a challenge in reconstructive surgery, especially with impaired healing potential due to severe trauma, infection or irradiation. In vivo studies are often performed in healthy animals, which might not accurately reflect the situation in clinical cases. In the present study, we successfully combined a critical-sized femoral defect model with an ionizing radiation protocol in rats. To support bone healing, tissue-engineered constructs were transferred into the defect after ectopic preossification and prevascularization. The combination of SiHA, MSCs and BMP-2 resulted in the significant ectopic formation of bone tissue, which can easily be transferred by means of our custom-made titanium chamber. Implanted osteogenic MSCs survived in vivo for a total of 18 weeks. The use of SiHA alone did not lead to bone formation after ectopic implantation. Analysis of gene expression showed early osteoblast differentiation and a hypoxic and inflammatory environment in implanted constructs. Irradiation led to impaired bone healing, decreased vascularization and lower short-term survival of implanted cells. We conclude that our model is highly valuable for the investigation of bone healing and tissue engineering in pre-damaged tissue and that healing of bone defects can be substantially supported by combining SiHA, MSCs and BMP-2.

Encapsulation of Mesenchymal Stem Cells Improves Vascularization of Alginate-Based Scaffolds.

Vascularization of bioartificial tissues can be significantly enhanced by the generation of an arteriovenous (AV) loop. Besides the surgical vascularization, the choice of the scaffold and the applied cells are indispensable cofactors. The combination of alginate dialdehyde and gelatin (ADA-GEL) and mesenchymal stem cells (MSCs) is a promising approach with regard to biocompatibility, biodegradation, as well as de novo tissue formation. In this study, we targeted the investigation of the vascularization of ADA-GEL with and in the absence of encapsulated MSCs in the AV loop model. A Teflon chamber filled with ADA-GEL microcapsules was placed in the groin of Lewis rats and an AV loop was placed into the chamber. Group A encompassed the ADA-GEL without MSCs, whereas group B contained 2 × 106 DiI-labeled MSCs/mL ADA-GEL. Four weeks postoperatively, tissue formation and vascularization were investigated by histology and microcomputed tomography. We were able to prove vascularization originating from the AV loop in both groups with statistically significant more vessels in group B containing MSCs. Moreover, encapsulated MSCs promoted biodegradation of the ADA-GEL microcapsules. In the present study, we were able to demonstrate for the first time, the successful vascularization of ADA-GEL microcapsules by means of the AV loop. Furthermore, ADA-GEL displayed a good biocompatibility and encapsulation of MSCs into ADA-GEL microcapsule-enhanced vascularization as well as biodegradation.

Encapsulation of Rat Bone Marrow Derived Mesenchymal Stem Cells in Alginate Dialdehyde/Gelatin Microbeads with and without Nanoscaled Bioactive Glass for In Vivo Bone Tissue Engineering.

Alginate dialdehyde (ADA), gelatin, and nano-scaled bioactive glass (nBG) particles are being currently investigated for their potential use as three-dimensional scaffolding materials for bone tissue engineering. ADA and gelatin provide a three-dimensional scaffold with properties supporting cell adhesion and proliferation. Combined with nanocristalline BG, this composition closely mimics the mineral phase of bone. In the present study, rat bone marrow derived mesenchymal stem cells (MSCs), commonly used as an osteogenic cell source, were evaluated after encapsulation into ADA-gelatin hydrogel with and without nBG. High cell survival was found in vitro for up to 28 days with or without addition of nBG assessed by calcein staining, proving the cell-friendly encapsulation process. After subcutaneous implantation into rats, survival was assessed by DAPI/TUNEL fluorescence staining. Hematoxylin-eosin staining and immunohistochemical staining for the macrophage marker ED1 (CD68) and the endothelial cell marker lectin were used to evaluate immune reaction and vascularization. After in vivo implantation, high cell survival was found after 1 week, with a notable decrease after 4 weeks. Immune reaction was very mild, proving the biocompatibility of the material. Angiogenesis in implanted constructs was significantly improved by cell encapsulation, compared to cell-free beads, as the implanted MSCs were able to attract endothelial cells. Constructs with nBG showed higher numbers of vital MSCs and lectin positive endothelial cells, thus showing a higher degree of angiogenesis, although this difference was not significant. These results support the use of ADA/gelatin/nBG as a scaffold and of MSCs as a source of osteogenic cells for bone tissue engineering. Future studies should however improve long term cell survival and focus on differentiation potential of encapsulated cells in vivo.

Influence of Different Irradiation Protocols on Vascularization and Bone Formation Parameters in Rat Femora.

Aim of the present study was the establishment of an efficient and reproducible model for irradiation of rat femora as a model for impaired osteogenesis and angiogenesis. Four different irradiation protocols were compared: single irradiation of the left femur with 20 Gy and explantation after 4 or 8 weeks (group A, B) and three irradiation fractions at 3-4 days intervals with 10 Gy and explantation after 4 or 8 weeks (group C, D). The contralateral, unirradiated femur served as control. Evaluation included histology, microcomputertomography (µCT), and real-time polymerase chain reaction. Histology showed a pronounced increase of vacuoles in bone marrow after irradiation, especially after 4 weeks (group A and C), demonstrating bone marrow edema and fatty degeneration. Irradiation provoked a decrease of total cell numbers in cortical bone and of hypoxia-inducible factor 1 alpha (HIF1α)-positive cells in bone marrow. The expression of several markers (osteocalcin [OCN], runt-related transcription factor 2 [RUNX2], transforming growth factor beta 1 [TGFß1], tumor necrosis factor alpha [TNFα], vascular endothelial growth factor A [VEGFA], and HIF1α) was decreased in group A after irradiation. This might suggest a decreased metabolism after irradiation. A significant decrease in small-sized vessels was seen in µCT evaluation in group A and D. Single irradiation with 20 Gy had the most severe and reproducible impact on osteogenesis and angiogenesis after 4 weeks while being well tolerated by all animals, thus making it an excellent model for evaluation of bone healing and vascularization in irradiated tissue.

Vascular Tissue Engineering: Effects of Integrating Collagen into a PCL Based Nanofiber Material.

The engineering of vascular grafts is a growing field in regenerative medicine. Although numerous attempts have been made, the current vascular grafts made of polyurethane (PU), Dacron®, or Teflon® still display unsatisfying results. Electrospinning of biopolymers and native proteins has been in the focus of research to imitate the extracellular matrix (ECM) of vessels to produce a small caliber, off-the-shelf tissue engineered vascular graft (TEVG) as a substitute for poorly performing PU, Dacron, or Teflon prostheses. Blended poly-ε-caprolactone (PCL)/collagen grafts have shown promising results regarding biomechanical and cell supporting features. In order to find a suitable PCL/collagen blend, we fabricated plane electrospun PCL scaffolds using various collagen type I concentrations ranging from 5% to 75%. We analyzed biocompatibility and morphological aspects in vitro. Our results show beneficial features of collagen I integration regarding cell viability and functionality, but also adverse effects like the loss of a confluent monolayer at high concentrations of collagen. Furthermore, electrospun PCL scaffolds containing 25% collagen I seem to be ideal for engineering vascular grafts.