Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept.

BACKGROUND: In vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-ß is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-ß can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN-212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-ß receptors during MSC in vitro chondrogenesis. METHODS: Standard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-ß and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice. RESULTS: All groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups. CONCLUSIONS: Overall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-ß that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-ß-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

[Platelet-rich plasma (PRP) : Compositional analysis with different dietary habits and timing of blood sampling]. / "Platelet-rich plasma" (PRP) : Analyse der Zusammensetzung bei unterschiedlichem Ernährungsverhalten und Blutentnahmezeitpunkt.

The variability of PRP is a major contributor to the lack of evidence regarding the therapeutic effect of PRP in musculoskeletal diseases. In a large study, we are currently investigating factors that may influence PRP variability. Interim results showed that concentrations of IL­6, but not IGF­1 or cellular constituents, were significantly decreased in PRP samples from vegans compared with omnivores and tended to be decreased compared to samples from vegetarians. This suggests that diet may have a significant influence on therapeutically active PRP constituents. However, the constituents studied here did not appear to be significantly affected by the timing of the sampling. Identification of significant variables affecting PRP composition will be critical to provide sufficient medical evidence for the therapeutic effects of PRP in orthopedic conditions.

Inverse Regulation of Cartilage Neogenesis at Physiologically Relevant Calcium Conditions by Human Articular Chondrocytes and Mesenchymal Stromal Cells.

Elaborate bioreactor cultivation or expensive growth factor supplementation can enhance extracellular matrix production in engineered neocartilage to provide sufficient mechanical resistance. We here investigated whether raising extracellular calcium levels in chondrogenic cultures to physiologically relevant levels would provide a simple and inexpensive alternative to enhance cartilage neogenesis from human articular chondrocytes (AC) or bone marrow-derived mesenchymal stromal cells (BMSC). Interestingly, AC and BMSC-derived chondrocytes showed an opposite response to a calcium increase from 1.8 mM to 8 mM by which glycosaminoglycan (GAG) and collagen type II production were elevated during BMSC chondrogenesis but depressed in AC, leading to two-fold higher GAG/DNA values in BMSC-based neocartilage compared to the AC group. According to control treatments with Mg2+ or sucrose, these effects were specific for CaCl2 rather than divalent cations or osmolarity. Importantly, undesired pro-hypertrophic traits were not stimulated by calcium treatment. Specific induction of PTHrP mRNA and protein by 8.0mM calcium only in AC, along with negative effects of recombinant PTHrP1-34 on cartilage matrix production, suggested that the PTHrP pathway contributed to the detrimental effects in AC-based neocartilage. Altogether, raising extracellular calcium levels was discovered as a novel, simple and inexpensive stimulator for BMSC-based cartilage neogenesis without the need for special bioreactors, whereas such conditions should be avoided for AC.

Adipose-Derived Stromal Cells: Isolation, Expansion, and Differentiation.

Adipose-derived stromal cells (ASC) are a promising alternative cell source to chondrocytes as well as to bone marrow-derived mesenchymal stromal cells (BMSC) in cartilage tissue engineering and repair. Here we describe ASC isolation from liposuction by-products by collagenase-based tissue digestion combined with cell filtration and followed by monolayer attachment and expansion culture. Quality control requires confirmation of correct surface marker expression and multilineage differentiation potential by a trilineage differentiation assay.

Stage-Dependent Activity and Pro-Chondrogenic Function of PI3K/AKT during Cartilage Neogenesis from Mesenchymal Stromal Cells.

Differentiating mesenchymal stromal cells (MSCs) into articular chondrocytes (ACs) for application in clinical cartilage regeneration requires a profound understanding of signaling pathways regulating stem cell chondrogenesis and hypertrophic degeneration. Classifying endochondral signals into drivers of chondrogenic speed versus hypertrophy, we here focused on insulin/insulin-like growth factor 1 (IGF1)-induced phosphoinositide 3-kinase (PI3K)/AKT signaling. Aware of its proliferative function during early but not late MSC chondrogenesis, we aimed to unravel the late pro-chondrogenic versus pro-hypertrophic PI3K/AKT role. PI3K/AKT activity in human MSC and AC chondrogenic 3D cultures was assessed via Western blot detection of phosphorylated AKT. The effects of PI3K inhibition with LY294002 on chondrogenesis and hypertrophy were assessed via histology, qPCR, the quantification of proteoglycans, and alkaline phosphatase activity. Being repressed by ACs, PI3K/AKT activity transiently rose in differentiating MSCs independent of TGFß or endogenous BMP/WNT activity and climaxed around day 21. PI3K/AKT inhibition from day 21 on equally reduced chondrocyte and hypertrophy markers. Proving important for TGFß-induced SMAD2 phosphorylation and SOX9 accumulation, PI3K/AKT activity was here identified as a required stage-dependent driver of chondrogenic speed but not of hypertrophy. Thus, future attempts to improve MSC chondrogenesis will depend on the adequate stimulation and upregulation of PI3K/AKT activity to generate high-quality cartilage from human MSCs.

NFκB inhibition to lift the mechano-competence of mesenchymal stromal cell-derived neocartilage toward articular chondrocyte levels.

BACKGROUND: Fully functional regeneration of skeletal defects by multipotent progenitor cells requires that differentiating cells gain the specific mechano-competence needed in the target tissue. Using cartilage neogenesis as an example, we asked whether proper phenotypic differentiation of mesenchymal stromal cells (MSC) into chondrocytes in vitro will install the adequate biological mechano-competence of native articular chondrocytes (AC). METHODS: The mechano-competence of human MSC- and AC-derived neocartilage was compared during differentiation for up to 35 days. The neocartilage layer was subjected to physiologic dynamic loading in a custom-designed bioreactor and assayed for mechano-sensitive gene and pathway activation, extracellular matrix (ECM) synthesis by radiolabel incorporation, nitric oxide (NO) and prostaglandin E2 (PGE2) production. Input from different pathways was tested by application of agonists or antagonists. RESULTS: MSC and AC formed neocartilage of similar proteoglycan content with a hardness close to native tissue. Mechano-stimulation on day 21 and 35 induced a similar upregulation of mechano-response genes, ERK phosphorylation, NO production and PGE2 release in both groups, indicating an overall similar transduction of external mechanical signals. However, while AC maintained or enhanced proteoglycan synthesis after loading dependent on tissue maturity, ECM synthesis was always significantly disturbed by loading in MSC-derived neocartilage. This was accompanied by significantly higher COX2 and BMP2 background expression, > 100-fold higher PGE2 production and a weaker SOX9 stimulation in response to loading in MSC-derived neocartilage. Anabolic BMP-pathway activity was not rate limiting for ECM synthesis after loading in both groups. However, NFκB activation mimicked the negative loading effects and enhanced PGE2 production while inhibition of catabolic NFκB signaling rescued the load-induced negative effects on ECM synthesis in MSC-derived neocartilage. CONCLUSIONS: MSC-derived chondrocytes showed a higher vulnerability to be disturbed by loading despite proper differentiation and did not acquire an AC-like mechano-competence to cope with the mechanical stress of a physiologic loading protocol. Managing catabolic NFκB influences was one important adaptation to install a mechano-resistance closer to AC-derived neocartilage. This new knowledge asks for a more functional adaptation of MSC chondrogenesis, novel pharmacologic co-treatment strategies for MSC-based clinical cartilage repair strategies and may aid a more rational design of physical rehabilitation therapy after AC- versus MSC-based surgical cartilage intervention.

The Role of Extracellular Matrix Expression, ERK1/2 Signaling and Cell Cohesiveness for Cartilage Yield from iPSCs.

Current therapies involving chondrocytes or mesenchymal stromal cells (MSCs) remain inefficient in restoring cartilage properties upon injury. The induced pluripotent stem-cell (iPSC)-derived mesenchymal progenitor cells (iMPCs) have been put forward as a promising alternative cell source due to their high proliferation and differentiation potential. However, the observed cell loss during in vitro chondrogenesis is currently a bottleneck in establishing articular chondrocyte generation from iPSCs. In a search for candidate mechanisms underlying the low iPSC-derived cartilage tissue yield, global transcriptomes were compared between iMPCs and MSCs and the cell properties were analyzed via a condensation assay. The iMPCs had a more juvenile mesenchymal gene signature than MSCs with less myofibroblast-like characteristics, including significantly lower ECM- and integrin-ligand-related as well as lower α-smooth-muscle-actin expression. This correlated with less substrate and more cell-cell adhesion, impaired aggregate formation and consequently inferior cohesive tissue properties of the iMPC-pellets. Along lower expression of pro-survival ECM molecules, like decorin, collagen VI, lumican and laminin, the iMPC populations had significantly less active ERK1/2 compared to MSCs. Overall, this study proposes that this ECM and integrin-ligand shortage, together with insufficient pro-survival ERK1/2-activity, explains the loss of a non-aggregating iMPC sub-fraction during pellet formation and reduced survival of cells in early pellets. Enhancing ECM production and related signaling in iMPCs may be a promising new means to enrich the instructive microenvironment with pro-survival cues allowing to improve the final cartilage tissue yield from iPSCs.

Regulation of WNT5A and WNT11 during MSC in vitro chondrogenesis: WNT inhibition lowers BMP and hedgehog activity, and reduces hypertrophy.

Re-directing mesenchymal stromal cell (MSC) chondrogenesis towards a non-hypertrophic articular chondrocyte-(AC)-like phenotype is important for improving articular cartilage neogenesis to enhance clinical cartilage repair strategies. This study is the first to demonstrate that high levels of non-canonical WNT5A followed by WNT11 and LEF1 discriminated MSC chondrogenesis from AC re-differentiation. Moreover, ß-catenin seemed incompletely silenced in differentiating MSCs, which altogether suggested a role for WNT signaling in hypertrophic MSC differentiation. WNT inhibition with the small molecule IWP-2 supported MSC chondrogenesis according to elevated proteoglycan deposition and reduced the characteristic upregulation of BMP4, BMP7 and their target ID1, as well as IHH and its target GLI1 observed during endochondral differentiation. Along with the pro-hypertrophic transcription factor MEF2C, multiple hypertrophic downstream targets including IBSP and alkaline phosphatase activity were reduced by IWP-2, demonstrating that WNT activity drives BMP and hedgehog upregulation, and MSC hypertrophy. WNT inhibition almost matched the strong anti-hypertrophic capacity of pulsed parathyroid hormone-related protein application, and both outperformed suppression of BMP signaling with dorsomorphin, which also reduced cartilage matrix deposition. Yet, hypertrophic marker expression under IWP-2 remained above AC level, and in vivo mineralization and ectopic bone formation were reduced but not eliminated. Overall, the strong anti-hypertrophic effects of IWP-2 involved inhibition but not silencing of pro-hypertrophic BMP and IHH pathways, and more advanced silencing of WNT activity as well as combined application of IHH or BMP antagonists should next be considered to install articular cartilage neogenesis from human MSCs.

StarPEG/heparin-hydrogel based in vivo engineering of stable bizonal cartilage with a calcified bottom layer.

Repaired cartilage tissue lacks the typical zonal structure of healthy native cartilage needed for appropriate function. Current grafts for treatment of full thickness cartilage defects focus primarily on a nonzonal design and this may be a reason why inferior nonzonal regeneration tissue developed in vivo. No biomaterial-based solutions have been developed so far to induce a proper zonal architecture into a non-mineralized and a calcified cartilage layer. The objective was to grow bizonal cartilage with a calcified cartilage bottom zone wherein main tissue development is occurring in vivo. We hypothesized that starPEG/heparin-hydrogel owing to the glycosaminoglycan heparin contained as a building-block would prevent mineralization of the upper cartilage zone and be beneficial in inhibiting long-term progression of calcified cartilage into bone. MSCs were pre-cultured as self-assembling non-mineralized cell discs before a chondrocyte-seeded fibrin- or starPEG/heparin-hydrogel layer was cast on top directly before ectopic implantation. Bizonal cartilage with a calcified bottom-layer developed in vivo showing stronger mineralization compared to in vitro samples, but the hydrogel strongly determined outcome. Zonal fibrin-constructs lost volume and allowed non-organized expansion of collagen type X, ALP-activity and mineralization from the bottom-layer into upper regions, whereas zonal starPEG/heparin-constructs were of stable architecture. While non-zonal MSCs-derived discs formed bone over 12 weeks, the starPEG/heparin-chondrocyte layer prevented further progression of calcified cartilage into bone tissue. Conclusively, starPEG/heparin-hydrogel-controlled and cell-type mediated spatiotemporal regulation allowed in vivo growth of bizonal cartilage with a stable calcified cartilage layer. Altogether our work is an important milestone encouraging direct in vivo growth of organized cartilage after biofabrication.

Stimulation of calvarial bone healing with human bone marrow stromal cells versus inhibition with adipose-tissue stromal cells on nanostructured ß-TCP-collagen.

Bioactive functional scaffolds are essential for support of cell-based strategies to improve bone regeneration. Adipose-tissue-derived-stromal cells (ASC) are more accessible multipotent cells with faster proliferation than bone-marrow-derived-stromal-cells (BMSC) having potential to replace BMSC for therapeutic stimulation of bone-defect healing. Their osteogenic potential is, however, lower compared to BMSC, a deficit that may be overcome in growth factor-rich orthotopic bone defects with enhanced bone-conductive scaffolds. Objective of this study was to compare the therapeutic potency of human ASC and BMSC for bone regeneration on a novel nanoparticulate ß-TCP/collagen-carrier (ß-TNC). Cytotoxicity of ß-TCP nanoparticles and multilineage differentiation of cells were characterized in vitro. Cell-seeded ß-TNC versus cell-free controls were implanted into 4 mm calvarial bone-defects in immunodeficient mice and bone healing was quantified by µCT at 4 and 8 weeks. Tissue-quality and cell-origin were assessed by histology. ß-TNC was non-toxic, radiolucent and biocompatible, lent excellent support for human cell persistence and allowed formation of human bone tissue by BMSC but not ASC. Opposite to BMSC, ASC-grafting significantly inhibited calvarial bone healing compared to controls. Bone formation progressed significantly from 4 to 8 weeks only in BMSC and controls yielding 5.6-fold more mineralized tissue in BMSC versus ASC-treated defects. Conclusively, ß-TNC was simple to generate, biocompatible, osteoconductive, and stimulated osteogenicity of BMSC to enhance calvarial defect healing while ASC had negative effects. Thus, an orthotopic environment and ß-TNC could not compensate for cell-autonomous deficits of ASC which should systematically be considered when choosing the right cell source for tissue engineering-based stimulation of bone regeneration. STATEMENT OF SIGNIFICANCE: Bone-marrow-derived-stromal cells (BMSC) implanted on bone replacement materials can support bone defect healing and adipose-tissue-derived-stromal cells (ASC) being more accessible and better proliferating are considered as alternate source. This first standardized comparison of the bone regeneration potency of human ASC and BMSC was performed on a novel nanoparticular ß-TCP-enriched collagen-carrier (ß-TNC) designed to overcome the known inferior osteogenicity of ASC. ß-TNC was non-toxic, biocompatible and osteoconductive supporting human bone formation and defect-closure by BMSC but not ASC. Long-term cell-persistence and the distinct secretome of ASC appear as main reasons why ASC inhibited bone healing opposite to BMSC. Overall, ASC-grafting is at considerable risk of producing negative effects on bone-healing while no such risks are known for BMSC.

Stimulation of a calcified cartilage connecting zone by GDF-5-augmented fibrin hydrogel in a novel layered ectopic in vivo model.

Tissue engineering approaches for reconstructing full-depth cartilage defects need to comprise a zone of calcified cartilage to tightly anchor cartilage constructs into the subchondral bone. Here, we investigated whether growth and differentiation factor-5-(GDF-5)-augmented fibrin hydrogel can induce a calcified cartilage-layer in vitro that seamlessly connects cartilage-relevant biomaterials with bone tissue. Human bone marrow stromal cells (BMSCs) were embedded in fibrin hydrogel and subjected to chondrogenesis with TGF-ß with or without GDF-5 before constructs were implanted subcutaneously into SCID mice. A novel layered ectopic in vivo model was developed and GDF-5-augmented fibrin with BMSCs was used to glue hydrogel and collagen constructs onto bone disks to investigate formation of a calcified cartilage connecting zone. GDF-5 significantly enhanced ALP activity during in vitro chondrogenesis while ACAN and COL2A1 mRNA, proteoglycan-, collagen-type-II- and collagen-type-X-deposition remained similar to controls. Pellets pretreated with GDF-5 mineralized faster in vivo and formed more ectopic bone. In the novel layered ectopic model, GDF-5 strongly supported calcified cartilage formation that seamlessly connected with the bone. Pro-chondrogenic and pro-hypertrophic activity makes GDF-5-augmented fibrin an attractive bioactive hydrogel with high potential to stimulate a calcified cartilage connecting zone in situ that might promote integration of cartilage scaffolds with bone. Thus, GDF-5-augmented fibrin hydrogel promises to overcome poor fixation of biomaterials in cartilage defects facilitating their long-term regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2214-2224, 2018.

Differential Regulation of SOX9 Protein During Chondrogenesis of Induced Pluripotent Stem Cells Versus Mesenchymal Stromal Cells: A Shortcoming for Cartilage Formation.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for cartilage regeneration, but current in vitro chondrogenic differentiation protocols yield insufficient results. In search for shortcomings of iPSC chondrogenesis, this study investigated whether SOX9 protein was adequately regulated during multiphase chondrogenic differentiation of two human iPSC lines in a comparable manner like during mesenchymal stromal cell (MSC) chondrogenesis. Upon generation of intermediate mesenchymal progenitor cells (iMPCs), SOX9 was induced and reached variable protein levels compared to MSCs. Along with an altered condensation behavior, iMPC cartilage formation was less robust compared to MSCs and better in the iMPC line with higher SOX9 protein levels. Despite efficient Smad-2/3 phosphorylation, TGF-ß-driven chondrogenic stimulation downregulated SOX9 protein in iMPCs rather than increasing levels like in MSCs. Chondrogenesis was further improved by cotreatment with TGF-ß + BMP-4, which appeared to shorten the duration of the SOX9 protein decline. However, this was insufficient to overcome heterogenic outcome and came at the expense of undesired hypertrophy. In iMPCs, but not MSCs, high levels of the SOX9-antagonizing hsa-miR-145 correlated with low SOX9 protein quantity. Thus, considerable iMPC heterogeneity with variable SOX9 protein levels, an altered condensation pattern, and low early SOX9 inducibility appeared as critical shortcomings of iPSC chondrogenesis. We suggest consistent quality of intermediate cell populations with high SOX9 protein induction as important indicators to obtain robust cartilage formation from iPSCs. The impact of this study is the identification of a SOX9 protein regulation opposite to MSC chondrogenesis that will now enable a selective adaptation of the currently limited protocols to the specific needs of iPSCs.

Xenogeneic transplantation of articular chondrocytes into full-thickness articular cartilage defects in minipigs: fate of cells and the role of macrophages.

Xenogeneic or allogeneic chondrocytes hold great potential to build up new cartilage in vivo. However, immune rejection is a major concern for the utility of universal donor-derived cells. In order to verify the reported immune privilege of chondrocytes in vivo, the aim of this study was to assess engraftment of human articular chondrocytes (HAC) in minipig knee cartilage defects and their contribution to cartilage regeneration. HAC were transplanted matrix-assisted within two hydrogels into full-thickness cartilage defects of minipigs or implanted ectopically into immune deficient mice to assess redifferentiation capacity. At 2 and 4 weeks after surgery, cell-persistence and host cell invasion were monitored by species-specific in situ hybridization and RT-PCR. Early tissue regeneration was evaluated by histomorphometry and a modified O'Driscoll score. HAC capable of successful in vivo chondrogenic redifferentiation persisted at ectopic sites for 4 weeks in both carrier materials. Early defect regeneration involved extensive host cell invasion and a decline of HAC to less than 5 % of initial cell numbers in 6/12 defects within 2 weeks. Few clusters of persisting HAC within collagen type II-rich tissue were surrounded by porcine macrophages. Four weeks after cell transplantation, most of the defects contained well-integrated cell-rich tissue free of human cells with no apparent difference between hydrogel carriers. In summary, HAC failed to engraft in porcine articular cartilage defects despite their ability for successful in vivo redifferentiation. The co-localization of macrophages to hydrogel-implanted HAC suggests active graft rejection without evidence for an immune-privileged status of xenogeneic chondrocytes in a large animal joint.

Regulating chondrogenesis of human mesenchymal stromal cells with a retinoic Acid receptor-Beta inhibitor: differential sensitivity of chondral versus osteochondral development.

AIM: Main objective was to investigate whether the synthetic retinoic acid receptor (RAR)-ß antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs) or improve differentiation by suppressing hypertrophic chondrocyte development. METHODS: Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-ß on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP), indian hedghehog (IHH) and matrix metalloproteinase (MMP)-13 were assessed. RESULTS: LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-ß-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. CONCLUSION: This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARß signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP)-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered.

Functional comparison of human-induced pluripotent stem cell-derived mesenchymal cells and bone marrow-derived mesenchymal stromal cells from the same donor.

Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases, and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are, thus, of interest, as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However, characterization of such MSC-like cells is currently inadequate, especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study, we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs), and compared the iMPCs directly with the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile, and they were capable of differentiation in vitro along the osteogenic, chondrogenic, and adipogenic lineages. However, compared with the parental BMSCs, iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We, therefore, conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics.

The promise and challenges of stem cell-based therapies for skeletal diseases: stem cell applications in skeletal medicine: potential, cell sources and characteristics, and challenges of clinical translation.

Despite decades of research, remaining safety concerns regarding disease transmission, heterotopic tissue formation, and tumorigenicity have kept stem cell-based therapies largely outside the standard-of-care for musculoskeletal medicine. Recent insights into trophic and immune regulatory activities of mesenchymal stem cells (MSCs), although incomplete, have stimulated a plethora of new clinical trials for indications far beyond simply supplying progenitors to replenish or re-build lost/damaged tissues. Cell banks are being established and cell-based products are in active clinical trials. Moreover, significant advances have also been made in the field of pluripotent stem cells, in particular the recent development of induced pluripotent stem cells. Their indefinite proliferation potential promises to overcome the limited supply of tissue-specific cells and adult stem cells. However, substantial hurdles related to their safety must be overcome for these cells to be clinically applicable.

Interplay between local versus soluble transforming growth factor-beta and fibrin scaffolds: role of cells and impact on human mesenchymal stem cell chondrogenesis.

Structural extracellular matrix molecules gain increasing attention as scaffolds for cartilage tissue engineering owing to their natural role as a growth factor repository. We recently observed that a collagen-type I/III (Col-I/III) matrix, human recombinant transforming growth factor-beta (TGF-ß) protein, and fibrin hydrogel (FG) combined to a biphasic construct provided sufficient long-term TGF-ß support to drive in vitro chondrogenesis of human mesenchymal stem cells (hMSC). Here we ask whether FG and Col-I/III can both retain TGF-ß, describe the influence of cell seeding on TGF-ß release, and compare the molecular path of hMSC chondrogenic differentiation under soluble versus local TGF-ß supply. Release of growth factor from scaffolds augmented with increasing amounts of TGF-ß was analyzed over 7 days and chondrogenesis was assessed over 42 days. Low TGF-ß release rates from Col-I/III as opposed to higher release from FG indicated that both molecules retained TGF-ß, with Col-I/III being the superior storage component. Cell seeding enhanced TGF-ß retention in FG by about threefold and almost stopped release beyond 24 h. TGF-ß remained bioactive and supported MSC chondrogenesis without impairing the amount of proteoglycan and collagen-type II deposition per cell and per construct compared to standard scaffold-free MSC pellets supplied with soluble TGF-ß. Local TGF-ß, however, mediated lower cell content, less collagen-type X relative to collagen-type II deposition and no matrix metalloproteinase-13 up-regulation. In conclusion, cells quickly halted release of local TGF-ß from FG, turning FG and Col-I/III into attractive TGF-ß repositories capable to drive full hMSC chondrogenesis, but via a modulated differentiation pathway. Since only part of the changes was reproduced by transient soluble TGF-ß supply, release kinetics alone could not explain the molecular differences, suggesting that local TGF-ß acts distinct from its soluble counterpart.

Application of different strain regimes in two-dimensional and three-dimensional adipose tissue-derived stem cell cultures induces osteogenesis: implications for bone tissue engineering.

Mechanical strain has become an important tool in tissue engineering for progenitor cell differentiation. Furthermore, it is used to enhance the mechanical properties of engineered tissue constructs. Although strain amplitude and frequency are well investigated and optimal values are known; application of various strain schemes regarding duration and repetition are not described in literature. In this study, we therefore applied singular and repetitive cyclic strain (1 Hz, 5%) of 15 min short-time strain and longer strain durations up to 8 h. Additionally, a gradually increasing strain scheme starting with short-time strain and consecutive elongated strain periods was applied. The cultivation surface was planar silicone on one hand and a three-dimensionally structured collagen I mesh on the other hand. Adipose tissue-derived mesenchymal stem cells and an osteogenic model cell line (MG-63) were exposed to these strain regimes and post-strain cell viability, osteogenic marker gene expression, and matrix mineralization were investigated. Upregulation of alkaline phosphatase, osteocalcin, osteopontin, and BMP-2/4 revealed that even short-time strain can enhance osteogenic differentiation. Elongation and repetition of strain, however, resulted in a decline of the observed short-time strain effects, which we interpret as positively induced cellular adaptation to the mechanically active surroundings. With regard to cellular adaptation, the gradually increasing strain scheme was especially advantageous.

Dynamic cultivation of human mesenchymal stem cells in a rotating bed bioreactor system based on the Z RP platform.

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three-dimensional functional tissue-like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three-dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram. Furthermore, hydroxyapatite coated Sponceram was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP-expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram compared to hydroxyapatite coated Sponceram. With this study, we present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components.

A study on the influence of biocompatible composites with bioactive ligands toward their effect on cell adhesion and growth for the application in bone tissue engineering.

The aim of this study was the transformation of the macroporous zirconium dioxide ceramic Sponceram into a biomimetic composite material. To enhance the adhesion of cells and to induce their differentiation into osteoblasts poly-L-lysine and BMP-2 were coupled to polymers and copolymers based on 2-deoxy-N-methacrylamido-D-glucose (ox.p(MAG) and p(MVA)) used as spacer, which were adsorbed onto the ceramic surface. The development of the composite materials was validated step by step qualitatively and quantitatively. The bioactive potential of the composite materials was tested under static and dynamic conditions using an osteoblastic model cell line and human mesenchymal stem cells. Both composite materials showed potential to enhance the adhesion of cells in the first 10 days of their cultivation. One of the composite materials, namely Sponceram/ox.p(MAG)-BMP-2, was tested into a rotating-bed bioreactor with regard to its osteogenic differentiation-inducing potential. Compared with Sponceram modified with BMP-2 without a polymer spacer, it showed increased expression of osteogenic markers determined by PCR analysis. In summary, the in vitro testing of the developed composite materials demonstrated a promising potential for their application as biomimetic scaffold materials with controllable properties.