Review of vascularised bone tissue‐engineering strategies with a focus on co‐culture systems

Poor angiogenesis within tissue‐engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue‐engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue‐engineered constructs and are briefly discussed in this review. Recent investigations relating to co‐culture systems of endothelial and osteoblast‐like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co‐culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co‐culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long‐lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.     

Multilineage co‐culture of adipose‐derived stem cells for tissue engineering

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue‐engineering strategies with adipose‐derived stem cells (ASCs), the focus of this study was on developing indirect co‐culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co‐culture with each of the mature cell populations was shown to successfully induce or enhance lineage‐specific differentiation of the ASCs. In general, a more homogeneous but lower‐level differentiation response was observed in co‐culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non‐canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF‐1 and DKK‐1 on multilineage differentiation in co‐culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co‐culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.     

Matrix formation is enhanced in co‐cultures of human meniscus cells with bone marrow stromal cells

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co‐cultured in vitro in three‐dimensional (3D) pellet culture at three different cell–cell ratios for 3 weeks under normal (21% O₂) or low (3% O₂) oxygen tension in the presence of serum‐free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT–PCR. Co‐cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3–1.7‐fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O₂. GAG matrix formation was also enhanced (1.3–1.6‐fold) under 3% O₂ culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG‐rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co‐cultured pellets. However, SOX9 and HIF‐1α mRNA expression were not significantly modulated by co‐culture. Co‐culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co‐delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Dynamic three‐dimensional micropatterned cell co‐cultures within photocurable and chemically degradable hydrogels

In this paper we report on the development of dynamically controlled three‐dimensional (3D) micropatterned cellular co‐cultures within photocurable and chemically degradable hydrogels. Specifically, we generated dynamic co‐cultures of micropatterned murine embryonic stem (mES) cells with human hepatocellular carcinoma (HepG2) cells within 3D hydrogels. HepG2 cells were used due to their ability to direct the differentiation of mES cells through secreted paracrine factors. To generate dynamic co‐cultures, mES cells were first encapsulated within micropatterned photocurable poly(ethylene glycol) (PEG) hydrogels. These micropatterned cell‐laden PEG hydrogels were subsequently surrounded by calcium alginate (Ca‐Alg) hydrogels containing HepG2 cells. After 4 days, the co‐culture step was halted by exposing the system to sodium citrate solution, which removed the alginate gels and the encapsulated HepG2 cells. The encapsulated mES cells were then maintained in the resulting cultures for 16 days and cardiac differentiation was analysed. We observed that the mES cells that were exposed to HepG2 cells in the co‐cultures generated cells with higher expression of cardiac genes and proteins, as well as increased spontaneous beating. Due to its ability to control the 3D microenvironment of cells in a spatially and temporally regulated manner, the method presented in this study is useful for a range of cell‐culture applications related to tissue engineering and regenerative medicine. Copyright © 2013 John Wiley & Sons, Ltd.     

Engineering cartilaginous grafts using chondrocyte‐laden hydrogels supported by a superficial layer of stem cells

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co‐cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self‐assembled on top of CC‐laden agarose gels (structured co‐culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co‐culture). Porcine infrapatellar fat pad‐derived stem cells (FPSCs) and bone marrow‐derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co‐culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co‐culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co‐culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co‐culture for both MSC sources. In conclusion, assembling MSCs onto CC‐laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro and in vivo co‐culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL–PEG–PCL hydrogels enhances cartilage formation

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co‐culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL–PEG–PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co‐cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co‐culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co‐culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co‐culture system in rabbit models. The co‐culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co‐culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Endothelial invasive response in a co‐culture model with physically‐induced osteodifferentiation

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co‐culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity‐induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co‐culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co‐culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen‐like morphology, suggesting that cell‐cell interaction in the co‐culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co‐culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre‐vascularized bone grafts. Copyright © 2012 John Wiley & Sons, Ltd.     

Adipose‐derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co‐culture with adipose‐derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co‐cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase‐14. Moreover, we showed OEC/ASC‐induced vessel‐like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co‐culture‐induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system. Copyright © 2012 John Wiley & Sons, Ltd.     

Low‐oxygen conditions promote synergistic increases in chondrogenesis during co‐culture of human osteoarthritic stem cells and chondrocytes

There has been increased interest in co‐cultures of stem cells and chondrocytes for cartilage tissue engineering as there are the limitations associated with using either cell type alone. Drawbacks associated with the use of chondrocytes include the limited numbers of cells available for isolation from damaged or diseased joints, their dedifferentiation during in vitro expansion, and a diminished capacity to synthesise cartilage‐specific extracellular matrix components with age and disease. This has motivated the use of adult stem cells with either freshly isolated or culture‐expanded chondrocytes for cartilage repair applications; however, the ideal combination of cells and environmental conditions for promoting robust chondrogenesis remains unclear. In this study, we compared the effect of combining a small number of freshly isolated or culture‐expanded human chondrocytes with infrapatellar fat pad–derived stem cells (FPSCs) from osteoarthritic donors on chondrogenesis in altered oxygen (5% or 20%) and growth factor supplementation (TGF‐β3 only or TGF‐β3 and BMP‐7) conditions. Both co‐cultures, but particularly those including freshly isolated chondrocytes, were found to promote cell proliferation and enhanced matrix accumulation compared to the use of FPSCs alone, resulting in the development of a tissue that was compositionally more similar to that of the native articular cartilage. Local oxygen levels were found to impact chondrogenesis in co‐cultures, with more robust increases in proteoglycan and collagen deposition observed at 5% O₂. Additionally, collagen type I synthesis was suppressed in co‐cultures maintained at low‐oxygen conditions. This study demonstrates that a co‐culture of freshly isolated human chondrocytes and FPSCs promotes robust chondrogenesis and thus is a promising cell combination for cartilage tissue engineering.     

Osteogenic capacity of human BM‐MSCs,AT‐MSCs and their co‐cultures using HUVECs in FBS and PL supplemented media

Human bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and human adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) are the most frequently used stem cells in tissue engineering. Due to major clinical demands, it is necessary to find an optimally safe and efficient way for large‐scale expansion of these cells. Considering the nutritional source in the culture medium and method, this study aimed to analyze the effects of FBS‐ and PL‐supplemented media on osteogenesis in stem cell mono‐ and co‐cultures with human umbilical vein endothelial cells (HUVECs). Results showed that cell metabolic activity and proliferation increased in PL‐ compared to FBS‐supplemented media in mono‐ and co‐cultures for both BM‐MSCs and AT‐MSCs. In addition, calcium deposition was cell type dependent and decreased for BM‐MSCs but increased for AT‐MSCs in PL‐supplemented medium in both mono‐ and co‐cultures. Based on the effects of co‐cultures, BM‐MSCs/HUVECs enhanced osteogenesis compared to BM‐MSCs monocultures in both FBS‐ and PL‐supplemented media whereas AT‐MSCs/HUVECs showed similar results compared to AT‐MSCs monocultures. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo construction of liver tissue by implantation of a hepatic non‐parenchymal/adipose‐derived stem cell sheet

Subcutaneous hepatocyte sheet implantation is an attractive therapeutic option for various liver diseases. However, this technique is limited by the availability of hepatocytes. Thus, the use of hepatic non‐parenchymal cells (NPCs) containing small hepatocytes, which have the ability to proliferate more rapidly than mature hepatocytes, for transplantation has been suggested. The aim of our study was to construct liver tissue subcutaneously in rats by implanting NPC sheets co‐cultivated with adipose‐derived stem cells (ADSCs), which produce certain angiogenic factors. We crafted NPC‐ADSC sheets on temperature‐responsive culture dishes. NPCs formed functioning bile canaliculi and stored glycogen. In addition, their ability to produce albumin was not inferior to that of hepatocytes. Albumin production increased over time when co‐cultivated with ADSCs. We then implanted the co‐cultivated cell sheets subcutaneously. The co‐cultivated sheets retained glycogen, formed bile canaliculi, showed signs of vascularization and survived subcutaneously without pre‐vascularization. These results suggest that NPCs can be a viable option in cell therapy for liver diseases. This technique using co‐cultivated cell sheets may be useful in the field of regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.     

Development of decellularized scaffolds for stem cell‐driven tissue engineering

Organ transplantation is an effective treatment for chronic organ dysfunctioning conditions. However, a dearth of available donor organs for transplantation leads to the death of numerous patients waiting for a suitable organ donor. The potential of decellularized scaffolds, derived from native tissues or organs in the form of scaffolds has been evolved as a promising approach in tissue‐regenerative medicine for translating functional organ replacements. In recent years, donor organs, such as heart, liver, lung and kidneys, have been reported to provide acellular extracellular matrix (ECM)‐based scaffolds through the process called ‘decellularization’ and proved to show the potential of recellularization with selected cell populations, particularly with stem cells. In fact, decellularized stem cell matrix (DSCM) has also emerged as a potent biological scaffold for controlling stem cell fate and function during tissue organization. Despite the proven potential of decellularized scaffolds in tissue engineering, the molecular mechanism responsible for stem cell interactions with decellularized scaffolds is still unclear. Stem cells interact with, and respond to, various signals/cues emanating from their ECM. The ability to harness the regenerative potential of stem cells via decellularized ECM‐based scaffolds has promising implications for tissue‐regenerative medicine. Keeping these points in view, this article reviews the current status of decellularized scaffolds for stem cells, with particular focus on: (a) concept and various methods of decellularization; (b) interaction of stem cells with decellularized scaffolds; (c) current recellularization strategies, with associated challenges; and (iv) applications of the decellularized scaffolds in stem cell‐driven tissue engineering and regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd.     

Micropatterned three‐dimensional hydrogel system to study human endothelial–mesenchymal stem cell interactions

The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell types and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (a) bone marrow aspirates; (b) perivascular cells from the umbilical cord vein; and (c) perivascular cells from the umbilical cord artery. Cell populations were isolated and identified as MSCs according to their phenotypes and differentiation potential. Human umbilical vein endothelial cells (HUVECs) were used as a standard for endothelial cells. A novel co‐culture system was developed to study cell–cell interactions in a spatially controlled three‐dimensional (3D) fibrin hydrogel model. Using microfluidic patterning, it was possible to localize hydrogel‐encapsulated HUVECs and MSCs within separate channels spaced at 500, 1000 or 2000 µm. All three MSC populations had similar expression profiles of mesenchymal cell markers and similar capacity for osteogenic and adipogenic differentiation. However, bone marrow‐derived MSCs (but not umbilical vein or artery derived MSCs) showed strong distance‐dependent migration toward HUVECs and supported the formation of stable vascular networks resembling capillary‐like vasculature. The presented approach provides a simple and robust model to study the cell–cell communication of relevance to engineering vascularized tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume‐persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self‐assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co‐culture spheroids combining adipose‐derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary‐like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT‐qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary‐like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high‐throughput (pre‐)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co‐culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular‐like structures within engineered tissues in vitro.     

Fabrication of tissue‐engineered cell sheets by automated cell culture equipment

Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.     

TGFβ3 secretion by three‐dimensional cultures of human dental apical papilla mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less‐studied MSC source. These dental‐derived MSCs are of great interest because of their potential as an accessible source for cell‐based therapies and tissue‐engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic‐mediated repair and regenerative effects observed when they are implanted. TGFβ3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP‐derived MSCs (hSCAPs) can produce and secrete TGFβ3 in response to micro‐environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFβ3 secretion. We found that dynamic changes of cell–matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two‐dimensional, 2D) towards a three‐dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFβ3 secretion, while monolayer cultures showed almost 10‐fold less secretion of TGFβ3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow‐derived MSCs in a co‐culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co‐culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co‐cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down‐regulated in co‐cultures on polystyrene, they were up‐regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co‐culture, direct cell‐cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Neutrophil‐mediated enhancement of angiogenesis and osteogenesis in a novel triple cell co‐culture model with endothelial cells and osteoblasts

Repair and regeneration of critical‐sized bone defects remain a major challenge in orthopaedic and craniomaxillofacial surgery. Until now, attempts to bioengineer bone tissue have been hindered by the inability to establish proper angiogenesis and osteogenesis in the tissue construct. In the present study, we established a novel triple cell co‐culture model consisting of osteoblasts, endothelial cells, and neutrophils and conducted a systematic investigation of the effects of neutrophils on angiogenesis and osteogenesis. Neutrophils significantly increased angiogenesis in the tissue construct, evidenced by the formation of microvessel‐like structures with an extensive lattice‐like, stable tubular network in the co‐culture model. Moreover, neutrophils significantly induced the expression of pro‐angiogenic markers, such as VEGF‐A, CD34, EGF, and FGF‐2 in a dose‐ and time‐dependent manner. Subsequently, PCR arrays corroborated that neutrophils upregulate the important angiogenic markers and MMPs. Moreover, neutrophils also enhanced osteogenic markers, such as ALP, OCN, OPN, and COL‐1 compared with the controls. As shown by the osteogenic gene arrays, neutrophils significantly regulated major osteogenic markers such as BMP2, BMP3, BMP4, BMP5, TGF‐β2, RUNX2, and ECM proteins. Significantly higher mineralization was observed in triple cell co‐culture compared with controls. Foregoing data indicate that the triple cell co‐culture model can be used to stimulate the growth of microvasculature within a bone bioengineering construct to improve cell viability. Neutrophil‐mediated enhancement of angiogenesis and osteogenesis could be a viable, clinically relevant tissue engineering strategy to obtain optimal bone growth in defect sites, in the field of oral and maxillofacial surgery.     

Co‐culture of adipose‐derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co‐implantation of adipose‐derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co‐cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell–fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co‐implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary‐like structures by co‐implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary‐like structures near the top of the construct, indicating that perfusion of the cell–fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.