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Autologous bone grafts are commonly used to repair defects in skeletal tissue, however, due to their limited supply there is a clinical need for alternatives. Synthetic ceramics present a promising option but currently lack biological activity to stimulate bone regeneration. One potential approach to address this limitation is the incorporation of immunomodulatory agents. In this study, we investigate the application of microbial stimuli to stimulate bone formation. Three different microbial stimuli were incorporated in a biphasic calcium phosphate (BCP) ceramic: Bacille Calmette-Guérin (BCG), gamma-irradiated Staphylococcus aureus ( γi- S. aureus) , or γi -Candida albicans (γi -C. Albicans ). The constructs were then implanted in both a rabbit posterolateral spinal fusion (PLF) and an intramuscular implant model for 10 weeks and compared to a nonstimulated control construct. For the PLF model, the formation of a bony bridge was evaluated by manual palpation, micro computed tomography, and histology. While complete fusion was not observed, the BCG condition was most promising with higher manual stiffness and almost twice as much bone volume in the central fusion mass compared to the control (9 ± 4.4% bone area vs. 4.6 ± 2.3%, respectively). Conversely, the γi- S. aureus or γi-C. albicans appeared to inhibit bone formation (1.4 ± 1.4% and 1.2 ± 0.6% bone area). Bone induction was not observed in any of the intramuscular implants. This study indicates that incorporating immunomodulatory agents in ceramic bone substitutes can affect bone formation, which can be positive when selected carefully. The readily available and clinically approved BCG showed promising results, which warrants further research for clinical translation.

Immune cells and their soluble factors have an important role in the bone healing process. Modulation of the immune response, therefore, offers a potential strategy to enhance bone formation. To investigate the influence of the immune system on osteogenesis, we developed and applied an in vitro model that incorporates both innate and adaptive immune cells. Human peripheral blood mononuclear cells (PBMCs) were isolated and cultured for 24 h and subsequently stimulated with immune-modulatory agents; C-class CpG oligodeoxynucleotide (CpG ODN C), polyinosinic acid-polycytidylic acid [Poly(I:C)], and lipopolysaccharide (LPS); all pathogen recognition receptor agonists, that target Toll-like receptors (TLRs) 9, 3, and 4, respectively. The conditioned medium (CM) obtained from PBMCs after 24 h was used to investigate its effects on the metabolic activity and osteogenic differentiation capacity of human bone marrow-derived mesenchymal stromal cells (MSCs). Conditioned media from unstimulated PBMCs did not affect the metabolic activity and osteogenic differentiation capacity of MSCs. The CM from CpG ODN C and LPS-stimulated PBMCs increased alkaline phosphatase activity (ALP) of MSCs by approximately threefold as compared with the unstimulated control, whereas Poly(I:C) CM did not enhance ALP activity of MSCs. Moreover, direct stimulation of MSCs with the immune-modulatory stimuli did not result in increased ALP. These results demonstrate that soluble factors present in CM from PBMCs stimulated with immune-modulatory factors enhance osteogenesis of MSCs. This in vitro model can serve as a tool in screening immune-modulatory stimulants from a broad variety of immune cells for (indirect) effects on osteogenesis and also to identify soluble factors from multiple immune cell types that may modulate bone healing.

Cardiovascular tissue engineering and regeneration strive to provide long-term, effective solutions for a growing group of patients in need of myocardial repair, vascular (access) grafts, heart valves, and regeneration of organ microcirculation. In the past two decades, ongoing convergence of disciplines and multidisciplinary collaborations between cardiothoracic surgeons, cardiologists, bioengineers, material scientists, and cell biologists have resulted in better understanding of the problems at hand and novel regenerative approaches. As a side effect, however, the field has become strongly organized and differentiated around topical areas at risk of reinvention of technologies and repetition of approaches across the areas. A better integration of knowledge and technologies from the individual topical areas and regenerative approaches and technologies may pave the way toward faster and more effective treatments to cure the cardiovascular system. This review summarizes the evolution of research and regenerative approaches in the areas of myocardial regeneration, heart valve and vascular tissue engineering, and regeneration of microcirculations; and discusses previous and potential future integration of these individual areas and developed technologies for improved clinical impact. Finally, it provides a perspective on the further integration of research organization, knowledge implementation, and valorization as a contributor to advancing cardiovascular tissue engineering and regenerative medicine. Impact Statement Despite ongoing convergence of disciplines, research in the field of cardiovascular tissue engineering and regeneration is organized and differentiated around focal areas, including myocardial regeneration, heart valve tissue engineering, vascular tissue engineering, and engineering of microcirculations. Cross-area integration of knowledge, supported by a more holistic, overarching research approach, may lead to faster and more effective treatments and prevent the reinvention of technologies across the areas. Herein, we review the evolution of research and technologies in the individual focal areas and discuss how to enhance integration of-and collaboration between-these areas for improved scientific and clinical impact.

Several urological structures, such as the male urethra, have a tubular organization consisting of different layers. However, in severe urethral disease, urologists are limited to replacing solely the epithelial layer. In case of severe hypospadias and urethral stricture disease, the underlying supporting structure (the corpus spongiosum) is either absent or fibrotic, causing suboptimal vascularization and therefore increasing the risk of graft failure. Recapitulating the multilayered architecture of the urethra, including supporting structure with tissue engineering, might minimize urethral graft failure. However, current tissue engineering applications for complex multilayered tubular constructs are limited. We describe a gel casting method to tissue engineer multilayered tubular constructs based on fiber-reinforced cell-laden hydrogels. For this, a multichambered polydimethylsiloxane mold was casted with fiber-reinforced hydrogels containing smooth muscle cells (SMCs) and a coculture of endothelial cells and pericytes. The cell-loaded hydrogels were rolled, with the fiber mesh as guidance, into a tubular construct. In the lumen, urothelial cells were seeded and survived for 2 weeks. In the tubular construct, the cells showed good viability and functionality: endothelial cells formed capillary-like structures supported by pericytes and SMCs expressed elastin. With a graft produced by this technique, supported with subepithelial vascularization, urethral reconstructive surgery can be improved. This approach toward tissue engineering of multilayered tubular structures can also be applied to other multilayered tubular structures found in the human body. Impact Statement Recapitulating the multilayered architecture of tubular structures found in the human body might minimize graft failure. Current tissue engineering applications for complex multilayered tubular constructs are limited. Here we describe a gel casting approach based on fiber-reinforced cell-laden hydrogels. A multichambered polydimethylsiloxane mold was casted with cell-loaded, fiber-reinforced hydrogels, with the fiber mesh as guidance, into a tubular construct. A graft produced by this technique can improve reconstructive surgery by providing subepithelial vascularization and thereby can reduce graft failure.

Pathologic conditions associated with bone formation can serve as models to identify bone-promoting mediators. The inflammatory response to bacterial infections generally leads to osteolysis and impaired bone healing, but paradoxically, it can also have pro-osteogenic effects. As a potential model to investigate pro-osteogenic stimuli, this study characterizes the bone formation in an established rabbit tibia model of periprosthetic infection. Our hypothesis was that the infection with Staphylococcus aureus (S. aureus) correlates with bone formation as a response to local inflammation. Fluorochromes showed excessive subperiosteal bone formation in infected tibiae, starting the first week and continuing throughout the study period. Despite the observed cortical lysis on micro-CT after 28 days, infection resulted in a twofold higher bone volume in the proximal tibiae compared to uninfected controls. The ipsilateral fibulae, nor the contralateral fibulae or tibiae were affected by infection. Next, we sought to confine the cause of stimulated bone formation to the isolated S. aureus cell wall. In absence of virulent bacterial infection, the S. aureus cell wall extract induced bone in a more favorable way without cortical lysis. This suggests that the sterile inflammatory reaction to bacterial antigens may be harnessed for bone regenerative purposes. Future investigations in this rabbit tibia model can lead to further identification of effective stimuli for clinical application.

Using a combination of articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) has shown to be a viable option for a single-stage cell-based treatment of focal cartilage defects. However, there is still considerable debate whether MSCs differentiate or have a chondroinductive role through trophic factors. In addition, it remains unclear whether direct cell-cell contact is necessary for chondrogenesis. Therefore, the aim of this study was to investigate whether direct or indirect cell-cell contact between ACs and MSCs is essential for increased cartilage production in different cellular environments and elucidate the mechanisms behind these cellular interactions. Human ACs and MSCs were cultured in a 10:90 ratio in alginate beads, fibrin scaffolds, and pellets. Cells were mixed in direct cocultures, separated by a Transwell filter (indirect cocultures), or cultured with conditioned medium. Short tandem repeat analysis revealed that the percentages of ACs increased during culture, while those of MSCs decreased, with the biggest change in fibrin glue scaffolds. For alginate, where the lack of cell-cell contact could be confirmed by histological analysis, no difference was found in matrix production between direct and indirect cocultures. For fibrin scaffolds and pellet cultures, an increased glycosaminoglycan production and type II collagen deposition were found in direct cocultures compared with indirect cocultures and conditioned medium. Positive connexin 43 staining and transfer of cytosolic calcein indicated communication through gap junctions in direct cocultures. Taken together, these results suggest that MSCs stimulate cartilage formation when placed in close proximity to chondrocytes and that direct cell-cell contact and communication through gap junctions are essential in this chondroinductive interplay.

Following an endochondral approach to bone regeneration, multipotent stromal cells (MSCs) can be cultured on a scaffold to create a cartilaginous callus that is subsequently remodeled into bone. An attractive scaffold material for cartilage regeneration that has recently regained attention is decellularized cartilage-derived matrix (CDM). Since this material has shown potential for cartilage regeneration, we hypothesized that CDM could be a potent material for endochondral bone regeneration. In addition, since decellularized matrices are known to harbor bioactive cues for tissue formation, we evaluated the need for seeded MSCs in CDM scaffolds. In this study, ectopic bone formation in rats was evaluated for CDM scaffolds seeded with human MSCs and compared with unseeded controls. The MSC-seeded samples were preconditioned in chondrogenic medium for 37 days. After 8 weeks of subcutaneous implantation, the extent of mineralization was significantly higher in the MSC-seeded constructs versus unseeded controls. The mineralized areas corresponded to bone formation with bone marrow cavities. In addition, rat-specific bone formation was confirmed by collagen type I immunohistochemistry. Finally, fluorochrome incorporation at 3 and 6 weeks revealed that the bone formation had an inwardly directed progression. Taken together, our results show that decellularized CDM is a promising biomaterial for endochondral bone regeneration when combined with MSCs at ectopic locations. Modification of current decellularization protocols may lead to enhanced functionality of CDM scaffolds, potentially offering the prospect of generation of cell-free off-the-shelf bone regenerative substitutes.

Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well as for endochondral bone tissue engineering, as these approaches deal with, respectively, the inhibition or enhancement of hypertrophic chondrogenesis.

To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, α-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1α was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1α expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.

Insufficient supply of oxygen and nutrients throughout the graft is considered one of the principal limitations in development of large, tissue-engineered bone grafts. Organ or tissue printing by means of three-dimensional (3D) fiber deposition is a novel modality in regenerative medicine that combines pore formation and defined cell placement, and is used here for development of cell-laden hydrogel structures with reproducible internal architecture to sustain oxygen supply and to support adequate tissue development. In this study we tested the effect of porosity on multipotent stromal cells (MSCs) embedded in hydrogel constructs printed with a 3D fiber deposition (3DF) machine. For this, porous and solid alginate hydrogel scaffolds, with MSCs homogeneously dispersed throughout the construct, were printed and analyzed in vitro for the presence of hypoxia markers, metabolism, survival, and osteogenic differentiation. We demonstrated that porosity promotes oxygenation of MSCs in printed hydrogel scaffolds and supported the viability and osteogenic differentiation of embedded cells. Porous and solid printed constructs were subsequently implanted subcutaneously in immunodeficient mice to analyze tissue formation in relation to hypoxia responses of embedded cells. Implantation of printed grafts resulted in ingrowth of vascularized tissue and significantly enhanced oxygenation of embedded MSCs. In conclusion, the introduction of pores significantly enhances the conductive properties of printed hydrogel constructs and contributes to the functionality of embedded osteogenic progenitors., (© Mary Ann Liebert, Inc.)

For years it has been recognized that engineering of large bone constructs will be feasible only if the hurdle of vascularization is overcome. Attempts to engineer bone tissue have predominantly focused on intramembranous (direct) bone formation. A relatively new and most likely more physiological approach in this line is endochondral bone formation, comprising an intermediate cartilaginous stage. Cartilage in nature is an avascular tissue and its cells are equipped to survive the poor oxygenation and nutritional conditions inherent to implanted tissues. Subsequent terminal differentiation (hypertrophy) of the chondrocytes initiates the formation of a mineralized matrix that will then be converted into bone. Through this mechanism, our long bones grow and most fractures heal through the process of secondary fracture healing. The feasibility of the attractive concept of endochondral bone tissue engineering has already been shown. Most emphasis has gone to the multipotent stromal cells because of their great potential for expansion and differentiation and immunoprivileged nature. This review will focus on the promises and current status of this new field. Further, potent modulators of endochondral bone tissue engineering, including oxygen tension and mechanical stimuli, will be discussed.

The influence of differentiation medium (DM) components on C2C12 murine myoblast differentiation has only been studied in monolayer cultures. Serum-free formulations have been applied that omit the use of sera with unknown composition. The goal of the present study was to compare the influence of serum-free media on C2C12 differentiation in 3-dimensional tissue-engineered muscle constructs. Myoblast proliferation and differentiation in media containing Ultroser G (DMU), insulin-like growth factor (IGF)-I (DMI), or both (DMUI) were compared with those induced by more-traditional media containing horse serum (HS) or horse serum and IGF-I (HSI). Effects of the applied media were assessed from gross construct morphology, total protein content, creatine kinase activity, and tissue viability. Addition of IGF-I (HSI) to the standard DM (HS) improved myoblast differentiation in muscle constructs. Even better results were obtained using DMU and DMUI culture conditions. DMI could not induce differentiation or maintain cell viability. Serum-free culture medium supplemented with DMU or DMUI accelerates and improves myoblast differentiation in engineered muscle tissue better than the gold standard HS.