Subha Narayan Rath

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three‐dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long‐term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform‐sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live‐dead assay, and real‐time RT‐PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell‐seeded scaffold product for applications in regenerative medicine.

Endothelial progenitor cells are integrated in newly formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a fibrin matrix.

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue‐engineered matrices. The aim of our study was to investigate the impact of a well‐characterized murine embryonal EPC line (T17b‐EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time‐points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing β‐galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell‐free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices.

Induction of bone formation in biphasic calcium phosphate scaffolds by bone morphogenetic protein-2 and primary osteoblasts

Bone tissue engineering strategies mainly depend on porous scaffold materials. In this study, novel biphasic calcium phosphate (BCP) matrices were generated by 3D‐printing. High porosity was achieved by starch consolidation. This study aimed to characterise the porous BCP‐scaffold properties and interactions of osteogenic cells and growth factors under in vivo conditions. Five differently treated constructs were implanted subcutaneously in syngeneic rats: plain BCP constructs (group A), constructs pre‐treated with BMP‐2 (group B; 1.6 µg BMP‐2 per scaffold), seeded with primary osteoblasts (OB) (group C), seeded with OB and BMP‐2 (group D) and constructs seeded with OB and pre‐cultivated in a flow bioreactor for 6 weeks (group E). After 2, 4 and 6 weeks, specimens were explanted and subjected to histological and molecular biological analyses. Explanted scaffolds were invaded by fibrovascular tissue without significant foreign body reactions. Morphometric analysis demonstrated significantly increased bone formation in samples from group D (OB + BMP‐2) compared to all other groups. Samples from groups B‐E displayed significant mRNA expression of bone‐specific genes after 6 weeks. Pre‐cultivation in the flow bioreactor (group E) induced bone formation comparable with group B. In this study, differences in bone distribution between samples with BMP‐2 or osteoblasts could be observed. In conclusion, combination of osteoblasts and BMP‐2 synergistically enhanced bone formation in novel ceramic scaffolds. These results provide the basis for further experiments in orthotopic defect models with a focus on future applications in orthopaedic and reconstructive surgery. Copyright

Foetal hepatocyte transplantation in a vascularized AV-Loop transplantation model in the rat

The use of foetal liver cells (FLC) in the context of hepatic tissue engineering might permit efficient in vitro expansion and cryopreservation in a cell bank. A prerequisite for successful application of bioartificial liver tissue is sufficient initial vascularization. In this study, we evaluated the transplantation of fibrin gel‐immobilized FLC in a vascularized arterio‐veno‐venous (AV)‐loop model. FLC were isolated from embryonic/foetal (ED 16) rat livers and were enriched by using magnetic cell sorting (MACS). After cryopreservation, FLC were labelled by pkh‐26. Cells were transplanted in a fibrin matrix into a subcutaneous chamber containing a microsurgically created AV‐loop in the femoral region of the recipient rat. The chambers were explanted after 14 days. Subcutaneous implants without an AV‐loop and cell‐free implants served as controls. Fluorescence microscopy of the constructs was used to identify pkh‐26+‐ donor cells. Characterization was performed by RT‐PCR and immunhistology (IH) for CK‐18 and CD31. Transplantation of FLC using the AV‐loop permitted a neo‐tissue formation in the fibrin matrix. A high‐density vascularization was observed in the AV‐loop constructs as shown by CD31 IH. Viable foetal donor cells were detected which expressed CK‐18. FLC can be successfully used for heterotopic transplantation. Fibrin matrix permits rapid blood vessel ingrowth from the AV‐loop and supports engraftment of FLC. It is therefore an appropriate environment for hepatocyte transplantation in combination with microsurgical vascularization strategies. Transplantation of fibrin gel‐immobilized FLC may be a promising approach for the development of highly vascularized in vivo tissue‐engineering‐based liver support systems.

Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches.

In vitro and in vivo Biocompatibility of Alginate Dialdehyde/Gelatin Hydrogels with and without Nanoscaled Bioactive Glass for Bone Tissue Engineering Applications

In addition to good mechanical properties needed for three-dimensional tissue engineering, the combination of alginate dialdehyde, gelatin and nano-scaled bioactive glass (45S5) is supposed to combine excellent cellular adhesion, proliferation and differentiation properties, good biocompatibility and predictable degradation rates. The goal of this study was to evaluate thein vitro and in vivo biocompatibility as a first step on the way to its use as a scaffold in bone tissue engineering. In vitro evaluation showed good cell adherence and proliferation of bone marrow derived mesenchymal stem cells seeded on covalently crosslinked alginate dialdehyde-gelatin (ADA-GEL) hydrogel films with and without 0.1% nano-Bioglass®(nBG). Lactate dehydrogenase (LDH)- and mitochondrial activity significantly increased in both ADA-GEL and ADA-GEL-nBG groups compared to alginate. However, addition of 0.1% nBG seemed to have slight cytotoxic effect compared to ADA-GEL. In vivo implantation did not produce a significant inflammatory reaction, and ongoing degradation could be seen after four weeks. Ongoing vascularization was detected after four weeks. The good biocompatibility encourages future studies using ADA-GEL and nBG for bone tissue engineering application.

In vitro evaluation of 45S5 Bioglass®‐derived glass‐ceramic scaffolds coated with carbon nanotubes

Highly porous (> 90% porosity) 45S5 Bioglass®-derived glass-ceramic scaffolds were fabricated by foam replication method, and coated with carbon nanotubes (CNT) (coating thickness: 1 μm) using electrophoretic deposition (EPD). In vitro cell culture using mesenchymal stem cells (MSCs) was carried out on both scaffold systems (with and without CNT coating) over a 4-week period. By using AlamarBlue™, BSA and alkaline phosphatase assays; the cell viability and differentiation were measured quantitatively measured and compared between the two scaffold types. The results showed that both scaffold systems are biocompatible with MSCs and they can support the cellular activity. No cytotoxic effects of CNT were observed under the conditions of the present experiments. Although a lower initial cell viability on the CNT-coated scaffolds was observed, no significant differences were found after 4 weeks of culture compared with the uncoated scaffolds. This work therefore shows that there is in principle no significant improvement of cellular responses by creating a CNT-coating on this type of highly bioactive scaffolds. However, the electrical conductivity introduced by the coating might have the potential to increase cell viability and differentiation when cell culture is carried out under the effect of electrical stimulation.

T17b murine embryonal endothelial progenitor cells can be induced towards both proliferation and differentiation in a fibrin matrix

Endothelial progenitor cells (EPC) may enhance blood vessel formation in a variety of clinical settings such as ischaemia and tumour angiogenesis as well as in tissue‐engineered matrices. In the present study, we cultured a murine endothelial progenitor cell line, T17b, in vitro in cell culture as well as in an FDA‐approved fibrin matrix and investigated cell proliferation, differentiation and secretion patterns of the angiogenic growth factor VEGF under hypoxia and differentiation. We show that T17b EPC remain viable for at least 8 days in the fibrin matrix where they proliferate and form clusters including lumen‐like structures. Proliferation in fibrin clots overlayed with basal medium (BM) was confirmed morphologically and immunohistochemically by positive Ki67 staining, indicating mitotic activity. Significant cell proliferation and Ki‐67 expression were absent when cells were incubated with dibutyryl‐cAMP and retinoic acid (RA). Incubation with dibutyryl‐cAMP and RA stimulated the expression of the EPC differentiation markers von Willebrand Factor (vWF) and VEGF receptor 2 (VEGFR‐2), indicating successful differentiation in the fibrin clot. EPC differentiation induced by dibutyryl‐cAMP and RA was confirmed in 2‐D chamber slide cultures by positive vWF immunostaining, which was absent in BM controls. EPC chamber slides also displayed positive vWF staining when exposed to hypoxia under BM conditions, indicating EPC activation and differentiation could also be induced by hypoxia. Taken together, T17b EPC secrete increased levels of VEGF when submitted to either hypoxia or differentiation and can be differentiated into mature endothelial cells not only in cell and matrigel cultures but also in a fibrin matrix that is FDA approved for clinical application.

Adipose- and bone marrow-derived mesenchymal stem cells display different osteogenic differentiation patterns in 3D bioactive glass-based scaffolds

Mesenchymal stem cells can be isolated from a variety of different sources, each having their own peculiar merits and drawbacks. Although a number of studies have been conducted comparing these stem cells for their osteo‐differentiation ability, these are mostly done in culture plastics. We have selected stem cells from either adipose tissue (ADSCs) or bone marrow (BMSCs) and studied their differentiation ability in highly porous three‐dimensional (3D) 45S5 Bioglass®‐based scaffolds. Equal numbers of cells were seeded onto 5 × 5 × 4 mm3 scaffolds and cultured in vitro, with or without osteo‐induction medium. After 2 and 4 weeks, the cell–scaffold constructs were analysed for cell number, cell spreading, viability, alkaline phosphatase activity and osteogenic gene expression. The scaffolds with ADSCs displayed osteo‐differentiation even without osteo‐induction medium; however, with osteo‐induction medium osteogenic differentiation was further increased. In contrast, the scaffolds with BMSCs showed no osteo‐differentiation without osteo‐induction medium; after application of osteo‐induction medium, osteo‐differentiation was confirmed, although lower than in scaffolds with ADSCs. In general, stem cells in 3D bioactive glass scaffolds differentiated better than cells in culture plastics with respect to their ALP content and osteogenic gene expression. In summary, 45S5 Bioglass‐based scaffolds seeded with ADSCs are well‐suited for possible bone tissue‐engineering applications. Induction of osteogenic differentiation appears unnecessary prior to implantation in this specific setting. Copyright

Comparison of chondrogenesis in static and dynamic environments using a SFF designed and fabricated PCL-PEO scaffold

A tssue engineering approach combining the scaffold/cell/matrix aspires to be a suitable alternative for current cartilage injury treatment modalities to produce a functional cartilage graft. Scaffolds synthesised from elastomeric materials and fabricated by a rapid prototyping technique were tested and investigated as a potential candidate for cartilage tissue engineering. The purpose of this study is to investigate the influence of scaffold internal architectures, matrix composition and dynamic environmental conditions on the final outcome of the scaffold-cell construct. PCL-PEO scaffolds were fabricated with 0°–90° and 0°–45° lay-down patterns with porosity values of 60% and 71% respectively. They were seeded with 500,000 porcine articular chondrocytes along with two different hydrogels, i.e. alginate and alginate/thrombin, and chondrocyte behaviour was studied. Furthermore, scaffold-cell constructs were cultured dynamically using the spinner flask method for 6 weeks. Porosity and surface-to-volume ratios were different for scaffolds with diverse geometry. The 0°–45° pattern scaffold showed significantly increased cell numbers and extracellular matrix proteins such as collagen-II compared to the 0°–90° scaffolds. The alginate/thrombin matrix encapsulated more chondrocytes compared to the alginate matrix alone. Cells were found to be viable and actively proliferating after 6 weeks of culture, maintaining the rounded chondrocyte phenotype in the matrix. Cell number, metabolic activity and extracellular matrix, such as collagen II, were produced significantly more in the dynamic environment compared to the static environment.