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Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, a conceptual shift based on the manufacturing of devitalized human hypertrophic cartilage (HyC), as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification, is reported. The strategy relies on a customized human mesenchymal line expressing bone morphogenetic protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization, and storage, the resulting off-the-shelf cartilage tissue exhibits unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy are demonstrated by bioreactor-based production and subsequent orthotopic assessment. The findings exemplify the broader paradigm of programming human cell lines as biological factory units to engineer customized ECMs, designed to activate specific regenerative processes.

Abstract Engineered and devitalized hypertrophic cartilage (HC) has been proposed as bone substitute material, potentially combining the features of osteoinductivity, resistance to hypoxia, capacity to attract blood vessels, and customization potential for specific indications. However, in comparison with vital tissues, devitalized HC grafts have reduced efficiency of bone formation and longer remodeling times. We tested the hypothesis that freshly harvested stromal vascular fraction (SVF) cells from human adipose tissue—which include mesenchymal, endothelial, and osteoclastic progenitors—enhance devitalized HC remodeling into bone tissue. Human SVF cells isolated from abdominal lipoaspirates were characterized cytofluorimetrically. HC pellets, previously generated by human bone marrow-derived stromal cells and devitalized by freeze/thaw, were embedded in fibrin gel with or without different amounts of SVF cells and implanted either ectopically in nude mice or in 4-mm-diameter calvarial defects in nude rats. In the ectopic model, SVF cells added to devitalized HC directly contributed to endothelial, osteoblastic, and osteoclastic populations. After 12 weeks, the extent of graft vascularization and amount of bone formation increased in a cell-number-dependent fashion (up to, respectively, 2.0-fold and 2.9-fold using 12 million cells per milliliter of gel). Mineralized tissue volume correlated with the number of implanted, SVF-derived endothelial cells (CD31+ CD34+ CD146+). In the calvarial model, SVF activation of HC using 12 million cells per milliliter of gel induced efficient merging among implanted pellets and strongly enhanced (7.3-fold) de novo bone tissue formation within the defects. Our findings outline a bone augmentation strategy based on off-the-shelf devitalized allogeneic HC, intraoperatively activated with autologous SVF cells. Significance This study validates an innovative bone substitute material based on allogeneic hypertrophic cartilage that is engineered, devitalized, stored, and clinically used, together with autologous cells, intraoperatively derived from a lipoaspirate. The strategy was tested using human cells in an ectopic model and an orthotopic implantation model, in immunocompromised animals.

Contains fulltext : 154670.pdf (Publisher’s version ) (Open Access) Nasal chondrocytes (NC) derive from the same multipotent embryological segment that gives rise to the majority of the maxillofacial bone and have been reported to differentiate into osteoblast-like cells in vitro. In this study, we assessed the capacity of adult human NC, appropriately primed towards hypertrophic or osteoblastic differentiation, to form bone tissue in vivo. Hypertrophic induction of NC-based micromass pellets formed mineralized cartilaginous tissues rich in type X collagen, but upon implantation into subcutaneous pockets of nude mice remained avascular and reverted to stable hyaline-cartilage. In the same ectopic environment, NC embedded into ceramic scaffolds and primed with osteogenic medium only sporadically formed intramembranous bone tissue. A clonal study could not demonstrate that the low bone formation efficiency was related to a possibly small proportion of cells competent to become fully functional osteoblasts. We next tested whether the cues present in an orthotopic environment could induce a more efficient direct osteoblastic transformation of NC. Using a nude rat calvarial defect model, we demonstrated that (i) NC directly participated in frank bone formation and (ii) the efficiency of survival and bone formation by NC was significantly higher than that of reference osteogenic cells, namely bone marrow-derived mesenchymal stromal cells. This study provides a proof-of-principle that NC have the plasticity to convert into bone cells and thereby represent an easily available cell source to be further investigated for craniofacial bone regeneration. 01 juni 2015

Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion.

In this study, we aimed at generating osteogenic and vasculogenic constructs starting from the stromal vascular fraction (SVF) of human adipose tissue as a single cell source. SVF cells from human lipoaspirates were seeded and cultured for 5 days in porous hydroxyapatite scaffolds by alternate perfusion through the scaffold pores, eliminating standard monolayer (two-dimensional [2D]) culture. The resulting cell-scaffold constructs were either enzymatically treated to extract and characterize the cells or subcutaneously implanted in nude mice for 8 weeks to assess the capacity to form bone tissue and blood vessels. SVF cells were also expanded in 2D culture for 5 days and statically loaded in the scaffolds. The SVF yielded 5.9 +/- 3.5 x 10(5) cells per milliliter of lipoaspirate containing both mesenchymal progenitors (5.2% +/- 0.9% fibroblastic colony forming units) and endothelial-lineage cells (54% +/- 6% CD34+/CD31+ cells). After 5 days, the total cell number was 1.8-fold higher in 2D than in three-dimensional (3D) cultures, but the percentage of mesenchymal- and endothelial-lineage cells was similar (i.e., 65%-72% of CD90+ cells and 7%-9% of CD34+/CD31+ cells). After implantation, constructs from both conditions contained blood vessels stained for human CD31 and CD34, functionally connected to the host vasculature. Importantly, constructs generated under 3D perfusion, and not those based on 2D-expanded cells, reproducibly formed bone tissue. In conclusion, direct perfusion of human adipose-derived cells through ceramic scaffolds establishes a 3D culture system for osteoprogenitor and endothelial cells and generates osteogenic-vasculogenic constructs. It remains to be tested whether the presence of endothelial cells accelerates construct vascularization and could thereby enhance implanted cell survival in larger size implants. Disclosure of potential conflicts of interest is found at the end of this article.

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose-derived cells. Copyright © 2015 John WileySons, Ltd.

In this study, we addressed whether Bone Sialoprotein (BSP) coating of various substrates could enhance the in vitro osteogenic differentiation and in vivo bone formation capacity of human Bone Marrow Stromal Cells (BMSC). Moreover, we tested whether synthetic polymer-based porous scaffolds, despite the absence of a mineral component, could support ectopic bone formation by human BMSC if coated with BSP. Adsorption of recombinant human BSP on tissue culture-treated polystyrene (TCTP), beta-tricalcium phosphate (Osteologic) or synthetic polymer (Polyactive) substrates was dose dependent, but did not consistently accelerate or enhance in vitro BMSC osteogenic differentiation, as assessed by the mRNA expression of osteoblast-related genes. Similarly, BSP coating of porous beta-tricalcium phosphate scaffolds (Skelite) did not improve the efficiency of bone tissue formation following loading with BMSC and ectopic implantation in nude mice. Finally, Polyactive foams seeded with BMSC did not form bone tissue in the same ectopic assay, even if coated with BSP. We conclude that BSP coating of a variety of substrates is not directly associated with an enhancement of osteoprogenitor cell differentiation in vitro or in vivo, and that presentation of BSP on polymeric materials is not sufficient to prime BMSC functional osteoblastic differentiation in vivo.

The connective tissue of the human tooth arises from cells that are derived from the cranial neural crest and, thus, are termed as "ectomesenchymal cells." Here, cells being located in a pad-like tissue adjacent to the apex of the developing tooth, which we designated the third molar pad, were separated by the microexplant technique. When outgrowing from the explant, dental neural crest-derived progenitor cells (dNC-PCs) adhered to plastic, proliferated steadily, and displayed a fibroblast-like morphology. At the mRNA level, dNC-PCs expressed neural crest marker genes like Sox9, Snail1, Snail2, Twist1, Msx2, and Dlx6. Cytofluorometric analysis indicated that cells were positive for CD49d (alpha4 integrin), CD56 (NCAM), and PDGFRalpha, while negative for CD31, CD34, CD45, and STRO-1. dNC-PCs could be differentiated into neurogenic, chondrogenic, and osteogenic lineages and were shown to produce bone matrix in athymic mice. These results demonstrate that human third molar pad possesses neural crest-derived cells that represent multipotent stem/progenitor cells. As a rather large amount of dNC-PCs could be obtained from each single third molar, cells may be used to regenerate a wide range of tissues within the craniofacial region of humans.

Biological substitutes for autologous bone flaps could be generated by combining flap pre-fabrication and bone tissue engineering concepts. Here, we investigated the pattern of neotissue formation within large pre-fabricated engineered bone flaps in rabbits. Bone marrow stromal cells from 12 New Zealand White rabbits were expanded and uniformly seeded in porous hydroxyapatite scaffolds (tapered cylinders, 10-20 mm diameter, 30 mm height) using a perfusion bioreactor. Autologous cell-scaffold constructs were wrapped in a panniculus carnosus flap, covered by a semipermeable membrane and ectopically implanted. Histological analysis, substantiated by magnetic resonance imaging (MRI) and micro-computerized tomography scans, indicated three distinct zones: an outer one, including bone tissue; a middle zone, formed by fibrous connective tissue; and a central zone, essentially necrotic. The depths of connective tissue and of bone ingrowth were consistent at different construct diameters and significantly increased from respectively 3.1 +/- 0.7 mm and 1.0 +/- 0.4 mm at 8 weeks to 3.7 +/- 0.6 mm and 1.4 +/- 0.6 mm at 12 weeks. Bone formation was found at a maximum depth of 1.8 mm after 12 weeks. Our findings indicate the feasibility of ectopic pre-fabrication of large cell-based engineered bone flaps and prompt for the implementation of strategies to improve construct vascularization, in order to possibly accelerate bone formation towards the core of the grafts.

Clinical experience indicates that the surface architecture of dental implants has an important impact on their integration. This has been related to the finding that differentially treated substrates can modulate the expression of osteogenic markers in various bone-related cell lines and primary cells. Here, we investigated the influence of surface architecture on the differentiation of human mesenchymal progenitor cells (HMPC) from adult bone marrow, i. e. the cells likely involved in initial bone synthesis at the bone-implant interface. Cells were seeded on machine surfaced (MS) or sandblasted/acid etched (SE) titanium discs in agarose-coated dishes, and on polystyrene (PS) controls. On all substrates cell densities did not change between days 7 and 14. Cell numbers were higher on SE, likely due to increased attachment to the rougher material. Alkaline phosphatase activity (ALP) was similar on all substrates, whereas mRNA expression of bone sialoprotein (BSP) at day 14 was about tenfold higher on SE (p0.05%). The SE-related increase of BSP in progenitor cells indicates an earlier differentiation of immigrated cells and could thus explain earlier implant integration and shorter time to functional loading observed in the clinic. The in vitro model and BSP quantification could be used to screen for changes in osteogenic cell differentiation induced by specific implant surfaces, with potential relevance on the prediction of bone-implant integration.

Congenital or acquired bony defects in the head and neck, as well as of long bones, can be treated using a variety of bone substitutes. If defects are small and surrounded by normal bone, materials from inorganic (ceramics), organic (polymers), or allogenic origin are typically used.1 Larger defects require either free autologous bone grafts or microvascular bone flaps harvested from different sites2. The most challenging problems associated with the latter techniques are the limited availability of free autologous bone grafts and the considerable donor site morbidity introduced by vascularized bone flaps.3 A promising approach to overcome these limitations is the use of osteogenic progenitor cells in combination with osteoconductive materials to engineer osteoinductive grafts. It is well known that bone marrow stroma contains progenitor cells with osteogenic potential, generally referred to as mesenchymal stem cells or bone marrow stromal cells (BMSCs).4–6 Human BMSCs have been demonstrated to differentiate toward the osteoblastic lineage in vitro,7,8 to form bone tissue upon ectopic implantation in nude mice,9 and to support the repair of large segmental defects in a few clinical cases.10 However, BMSCs are found in human marrow aspirates at low frequency (approximately 0.01% of the total mononucleated cells), and their expansion by serial passaging, which is consequently required, is associated with a dramatic reduction in the differentiation capacity.11,12 In addition, a wide variability has been reported in the osteogenic ability of BMSCs from different donors13,14 or harvested using different procedures.15 Moreover, the aspiration of bone marrow has to be carried out in a sterile environment and is generally associated with considerable pain. It is also well established that the periosteum is a source of cells with osteogenic potential, which is maintained even in elderly individuals.16 Human periosteal cells loaded into porous polymeric scaffolds have been shown to differentiate toward the osteoblastic lineage in vitro17 and to form traces of bone tissue ectopically.18 Harvest of the jaw periosteum as a source of osteogenic cells is particularly appealing because of the simplicity of the procedure and the minimal morbidity induced. Recently, Schimming and Schmelzeisen reported the clinical use of jaw periosteal cells (JPCs) in combination with a polymer fleece in the context of the maxillary sinus elevation procedure.19 Although radiologically detectable bone formation was found in 18 of 27 patients, it was not possible to prove that the newly deposited bone tissue was formed by the implanted periosteal cells and not by cells surrounding the defect. Thus, it remains unclear to which extent human JPCs are capable to induce bone formation in vivo and how this capacity is related to that of BMSCs. In this paper, we first aimed at comparing the in vitro differentiation and in vivo bone formation of BMSCs and JPCs from the same individuals, using 2-dimensional (2D) and 3-dimensional (3D) culture models and an ectopic assay in nude mice. Considering the need of reproducibility or at least of predictability in the cell osteoinductive ability for a standardized clinical use, we then investigated whether the patterns of expression of osteoblast-specific markers in 2D or 3D cultures could be predictive of the cell capacity to form bone tissue in the ectopic model.

Background Bone tissue formation by bone marrow stromal cells may be supported and enhanced by multiple growth factors, particularly in cases of a compromised local microenvironment. In this study, the authors hypothesized that fibroblast growth factor (FGF)-2 can stimulate the production by human bone marrow stromal cells of osteogenic [i.e., bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1] and angiogenic [i.e., vascular endothelial growth factor (VEGF)] factors. Methods Human bone marrow stromal cells from six donors were expanded for two passages (expansion phase) and subsequently cultivated in osteogenic medium containing ascorbic acid, beta-glycerophosphate, and dexamethasone (differentiation phase). After each phase, cells were transferred into serum-free medium with or without FGF-2 at different concentrations and for different times, and the expression of BMP-2, TGF-beta1, and VEGF was quantified at the mRNA level by real-time quantitative reverse-transcriptase polymerase chain reaction. The amounts of TGF-beta1 and VEGF released in the culture medium were assessed using enzyme-linked immunosorbent assay kits and normalized to the DNA content. Results In response to 5 ng/ml FGF-2 for 24 hours, the mRNA expression of VEGF increased at both culture phases (up to 6.1 fold), whereas that of BMP-2 and TGF-beta1 significantly increased only after the expansion (3.1-fold) or differentiation phase (2.1-fold), respectively. Similar trends were observed in the amounts of proteins measured in the culture medium. Conclusions The authors' results indicate that FGF-2 up-regulates the expression of BMP-2, TGF-beta1, and VEGF in human bone marrow stromal cells, in a pattern dependent on the cell-differentiation stage. These findings prompt for in vivo investigations on the delivery of FGF-2 for the temporally/functionally regulated enhancement of bone marrow stromal cell-based bone induction.

Three-dimensional (3D) culture systems are critical to investigate cell physiology and to engineer tissue grafts. In this study, we describe a simple yet innovative bioreactor-based approach to seed, expand, and differentiate bone marrow stromal cells (BMSCs) directly in a 3D environment, bypassing the conventional process of monolayer (two-dimensional [2D]) expansion. The system, based on the perfusion of bone marrow-nucleated cells through porous 3D scaffolds, supported the formation of stromal-like tissues, where BMSCs could be cocultured with hematopoietic progenitor cells in proportions dependent on the specific medium supplements. The resulting engineered constructs, when implanted ectopically in nude mice, generated bone tissue more reproducibly, uniformly, and extensively than scaffolds loaded with 2D-expanded BMSCs. The developed system may thus be used as a 3D in vitro model of bone marrow to study interactions between BMSCs and hematopoietic cells as well as to streamline manufacture of osteoinductive grafts in the context of regenerative medicine.