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.

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.

MiR-218 affects hypertrophic differentiation of human mesenchymal stromal cells during chondrogenesis via targeting RUNX2, MEF2C, and COL10A1.

BACKGROUND: Human mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. However, undesirable occurrence of calcification after ectopic transplantation, known as hypertrophic degeneration, remains the major obstacle limiting application of MSC in cartilage tissue regeneration approaches. There is growing evidence that microRNAs (miRs) play essential roles in post-transcriptional regulation of hypertrophic differentiation during chondrogenesis. Aim of the study was to identify new miR candidates involved in repression of hypertrophy-related targets. METHODS: The miR expression profile in human articular chondrocytes (AC) was compared to that in hypertrophic chondrocytes derived from human MSC by microarray analysis, and miR expression was validated by qPCR. Putative targets were searched by in silico analysis and validated by miR reporter assay in HEK293T, by functional assays (western blotting and ALP-activity) in transiently transfected SaOS-2 cells, and by a miR pulldown assay in human MSC. The expression profile of miR-218 was assessed by qPCR during in vitro chondrogenesis of MSC and re-differentiation of AC. MSC were transfected with miR-218 mimic, and differentiation outcome was assessed over 28 days. MiR-218 expression was quantified in healthy and osteoarthritic cartilage of patients. RESULTS: Within the top 15 miRs differentially expressed between chondral AC versus endochondral MSC differentiation, miR-218 was selected as a candidate miR predicted to target hypertrophy-related genes. MiR-218 was downregulated during chondrogenesis of MSC and showed a negative correlation to hypertrophic markers, such as COL10A1 and MEF2C. It was confirmed in SaOS-2 cells that miR-218 directly targets hypertrophy-related COL10A1, MEF2C, and RUNX2, as a gain of ectopic miR-218 mimic caused drop in MEF2C and RUNX2 protein accumulation, with attenuation of COL10A1 expression and significant concomitant reduction of ALP activity. A miR pulldown assay confirmed that miR-218 directly targets RUNX2, MEF2C in human MSC. Additionally, the gain of miR-218 in human MSC attenuated hypertrophic markers (MEF2C, RUNX2, COL10A1, ALPL), although with no boost of chondrogenic markers (GAG deposition, COL2A1) due to activation of WNT/ß-catenin signaling. Moreover, no correlation between miR-218 expression and a pathologic phenotype in the cartilage of osteoarthritis (OA) patients was found. CONCLUSIONS: Although miR-218 was shown to target pro-hypertrophic markers MEF2C, COL10A1, and RUNX2 in human MSC during chondrogenic differentiation, overall, it could not significantly reduce the hypertrophic phenotype or boost chondrogenesis. This could be explained by a concomitant activation of WNT/ß-catenin signaling counteracting the anti-hypertrophic effects of miR-218. Therefore, to achieve a full inhibition of the endochondral pathway, a whole class of anti-hypertrophic miRs, including miR-218, needs to be taken into consideration.

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.

Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo. METHODS: MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated. RESULTS: C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice. CONCLUSIONS: C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion.

Treatment of Focal Cartilage Defects in Minipigs with Zonal Chondrocyte/Mesenchymal Progenitor Cell Constructs.

Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (µCT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O'Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage.

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.

Time-dependent contribution of BMP, FGF, IGF, and HH signaling to the proliferation of mesenchymal stroma cells during chondrogenesis.

Early loss of up to 50% of cells is common for in vitro chondrogenesis of mesenchymal stromal cells (MSC) in pellet culture, reducing the efficacy and the tissue yield for cartilage engineering. Enhanced proliferation could compensate for this unwanted effect, but relevant signaling pathways remain largely unknown. The aim of this study was to identify the contribution of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and hedgehog (HH) signaling toward cell proliferation during chondrogenesis and investigate whether a further mitogenic stimulation is possible and promising. Human MSC were subjected to chondrogenesis in the presence or absence of pathway inhibitors or activators up to Day 14 or from Days 14 to 28, before proliferation, DNA and proteoglycan content were quantified. [3H]-thymidine incorporation revealed arrest of proliferation on Day 3, after which cell division was reinitiated. Although BMP signaling was essential for proliferation throughout chondrogenesis, IGF signaling was relevant only up to Day 14. In contrast, FGF and HH signaling drove proliferation only from Day 14 onward. Early BMP4, IGF-1, or FGF18 treatment neither prevented early cell loss nor allowed further mitogenic stimulation. However, application of the HH-agonist purmorphamine from Day 14 increased proliferation 1.44-fold (p < 0.05) and late BMP4-application enhanced the DNA and proteoglycan content, with significant effects on tissue yield. Conclusively, a differential and phase-dependent contribution of the four pathways toward proliferation was uncovered and BMP4 treatment was promising to enhance tissue yield. Culture forms less prone to size limitations by nutrient/oxygen gradients and a focus on early apoptosis prevention may be considered as the next steps to further enhance chondrocyte formation from MSC.

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.

The Radiation Resistance of Human Multipotent Mesenchymal Stromal Cells Is Independent of Their Tissue of Origin.

PURPOSE: Human mesenchymal stromal cells (MSCs) may aid the regeneration of ionizing radiation (IR)-induced tissue damage. They can be harvested from different tissues for clinical purposes; however, the role of the tissue source on the radiation response of human MSCs remains unknown. METHODS AND MATERIALS: Human MSCs were isolated from adipose tissue, bone marrow, and umbilical cord, and cellular survival, proliferation, and apoptosis were measured after irradiation. The influence of IR on the defining functions of MSCs was assessed, and cell morphology, surface marker expression, and the differentiation potential were examined. Western blot analyses were performed to assess the activation of DNA damage signaling and repair pathways. RESULTS: MSCs from adipose tissue, bone marrow, and umbilical cord exhibited a relative radioresistance independent of their tissue of origin. Defining properties including cellular adhesion and surface marker expression were preserved, and irradiated MSCs maintained their potential for multilineage differentiation irrespective of their tissue source. Analysis of activated DNA damage recognition and repair pathways demonstrated an efficient repair of IR-induced DNA double-strand breaks in MSCs from different tissues, thereby influencing the induction of apoptosis. CONCLUSIONS: These data show for the first time that MSCs are resistant to IR and largely preserve their defining functions after irradiation irrespective of their tissue of origin. Efficient repair of IR-induced DNA double-strand breaks and consecutive reduction of apoptosis induction may contribute to the tissue-independent radiation resistance of MSCs.

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.

Peptide-functionalized starPEG/heparin hydrogels direct mitogenicity, cell morphology and cartilage matrix distribution in vitro and in vivo.

Cell-based tissue engineering is a promising approach for treating cartilage lesions, but available strategies still provide a distinct composition of the extracellular matrix and an inferior mechanical property compared to native cartilage. To achieve fully functional tissue replacement more rationally designed biomaterials may be needed, introducing bioactive molecules which modulate cell behavior and guide tissue regeneration. This study aimed at exploring the impact of cell-instructive, adhesion-binding (GCWGGRGDSP called RGD) and collagen-binding (CKLER/CWYRGRL) peptides, incorporated in a tunable, matrixmetalloprotease (MMP)-responsive multi-arm poly(ethylene glycol) (starPEG)/heparin hydrogel on cartilage regeneration parameters in vitro and in vivo. MMP-responsive-starPEG-conjugates with cysteine termini and heparin-maleimide, optionally pre-functionalized with RGD, CKLER, CWYRGRL or control peptides, were cross-linked by Michael type addition to embed and grow mesenchymal stromal cells (MSC) or chondrocytes. While starPEG/heparin-hydrogel strongly supported chondrogenesis of MSC according to COL2A1, BGN and ACAN induction, MMP-degradability enhanced cell viability and proliferation. RGD-modification of the gels promoted cell spreading with intense cell network formation without negative effects on chondrogenesis. However, CKLER and CWYRGRL were unable to enhance the collagen content of constructs. RGD-modification allowed more even collagen type II distribution by chondrocytes throughout the MMP-responsive constructs, especially in vivo. Collectively, peptide-instruction via heparin-enriched MMP-degradable starPEG allowed adjustment of self-renewal, cell morphology and cartilage matrix distribution in order to guide MSC and chondrocyte-based cartilage regeneration towards an improved outcome. Copyright © 2017 John Wiley & Sons, Ltd.

Is Total Disk Replacement a Cost-effective Treatment for Cervical Degenerative Disk Disease?

STUDY DESIGN: A retrospective study. OBJECTIVE: The objective of this study was to assess the cost-effectiveness and clinical outcome of motion-preserving versus fusion procedures in cervical spine surgery. SUMMARY OF BACKGROUND DATA: During the last decade there has been a huge growth in spine surgery with a concurrent increase in the economic burden. Currently, there appear to be no differences in clinical outcome between cervical total disk replacement (TDR) and spinal fusion (SF). For this reason it seems useful to know within the decision-making process whether there is a difference in actual cost between motion-preserving and fusion surgery. So far data that describe expenses involved in these procedures have not been available. This study offers a comparison of economic factors that should be considered in TDR and SF. MATERIALS AND METHODS: The German statutory general healthcare insurance (GHI) provides anonymized patient-related data of their customers. A retrospective query using the codes of surgery of all TDR and SF surgery was performed from January 2003 to June 2008. A total of 467 cases with monosegmental or bisegmental surgery for degenerative disk pathologies were included. RESULTS: Both groups showed significant differences in independent variables such as age and sex (P<0.0001), but not in revision rates. Cost weight of diagnosis-related groups and length of hospitalization had a significant effect on total costs. Both groups obtained less pain medication postoperatively than preoperatively without a significant difference between each group. Postoperative absenteeism from work was significantly higher in the TDR group;however, patients with TDR underwent less rehabilitation covered by the GHI. Both groups had the same amount of preoperative and postoperative physiotherapy covered by the GHI. CONCLUSIONS: According to the collected data, there are no differences between the medical outcomes of cervical TDR in comparison with cervical SF. At the same time, while generating clinical results comparable with spinal fusion, TDR incurred significantly lower costs. Therefore, both from the medical and from the financial point of view, TDR is a viable choice in the treatment of degenerative disk pathology.

Osteoarthritis in the Knee Joints of Göttingen Minipigs after Resection of the Anterior Cruciate Ligament? Missing Correlation of MRI, Gene and Protein Expression with Histological Scoring.

INTRODUCTION: The Göttingen Minipig (GM) is used as large animal model in articular cartilage research. The aim of the study was to introduce osteoarthritis (OA) in the GM by resecting the anterior cruciate ligament (ACLR) according to Pond and Nuki, verified by histological and magnetic resonance imaging (MRI) scoring as well as analysis of gene and protein expression. MATERIALS AND METHODS: The eight included skeletally mature female GM were assessed after ACLR in the left and a sham operation in the right knee, which served as control. 26 weeks after surgery the knee joints were scanned using a 3-Tesla high-field MR tomography unit with a 3 T CP Large Flex Coil. Standard proton-density weighted fat saturated sequences in coronal and sagittal direction with a slice thickness of 3 mm were used. The MRI scans were assessed by two radiologists according to a modified WORMS-score, the X-rays of the knee joints by two evaluators. Osteochondral plugs with a diameter of 4mm were taken for histological examination from either the main loading zone or the macroscopic most degenerated parts of the tibia plateau or condyle respectively. The histological sections were blinded and scored by three experts according to Little et al. Gene expression analysis was performed from surrounding cartilage. Expression of adamts4, adamts5, acan, col1A1, col2, il-1ß, mmp1, mmp3, mmp13, vegf was determined by qRT-PCR. Immunohistochemical staining (IH) of Col I and II was performed. IH was scored using a 4 point grading (0-no staining; 3-intense staining). RESULTS AND DISCUSSION: Similar signs of OA were evident both in ACLR and sham operated knee joints with the histological scoring result of the ACLR joints with 6.48 ± 5.67 points and the sham joints with 6.86 ± 5.84 points (p = 0.7953) The MRI scoring yielded 0.34 ± 0.89 points for the ACLR and 0.03 ± 0.17 for the sham knee joints. There was no correlation between the histological and MRI scores (r = 0.10021). The gene expression profiles as well as the immunohistochemical findings showed no significant differences between ACLR and sham knee joints. In conclusion, both knee joints showed histological signs of OA after 26 weeks irrespective of whether the ACL was resected or not. As MRI results did not match the histological findings, MRI was obviously unsuitable to diagnose the OA in GM. The analysis of the expression patterns of the 10 genes could not shed light on the question, whether sham operation also induced cartilage erosion or if the degeneration was spontaneous. The modified Pond-Nuki model may be used with reservation in the adult minipig to induce an isolated osteoarthritis.

Differential expression of TGF-ß superfamily members and role of Smad1/5/9-signalling in chondral versus endochondral chondrocyte differentiation.

Proteins of the transforming-growth-factor-ß (TGF-ß)-superfamily have a remarkable ability to induce cartilage and bone and the crosstalk of TGF-ß - and BMP-signalling pathways appears crucial during chondrocyte development. Aim was to assess the regulation of TGF-ß-superfamily members and of Smad2/3- and Smad1/5/9-signalling during endochondral in vitro chondrogenesis of mesenchymal stromal cells (MSC) relative to chondral redifferentiation of articular chondrocytes (AC) to adjust chondrocyte development of MSC towards a less hypertrophic phenotype. While MSC increased BMP4 and BMP7 and reduced TGFBR2 and TGFBR3-expression during chondrogenesis, an opposite regulation was observed during AC-redifferentiation. Antagonists CHRD and CHL2 rose significantly only in AC-cultures. AC showed higher initial BMP4, pSmad1/5/9 and SOX9 protein levels, a faster (re-)differentiation but a similar decline of pSmad2/3- and pSmad1/5/9-signalling versus MSC-cultures. BMP-4/7-stimulation of MSC-pellets enhanced SOX9 and accelerated ALP-induction but did not shift differentiation towards osteogenesis. Inhibition of BMP-signalling by dorsomorphin significantly reduced SOX9, raised RUNX2, maintained collagen-type-II and collagen-type-X lower and kept ALP-activity at levels reached at initiation of treatment. Conclusively, ALK1,2,3,6-signalling was essential for MSC-chondrogenesis and its prochondrogenic rather than prohypertrophic role may explain why inhibition of canonical BMP-signalling could not uncouple cartilage matrix production from hypertrophy as this was achieved with pulsed PTHrP-application.

Role of PTHrP(1-34) Pulse Frequency Versus Pulse Duration to Enhance Mesenchymal Stromal Cell Chondrogenesis.

Generation of phenotypically stable, articular chondrocytes from mesenchymal stromal cells (MSCs) is still an unaccomplished task, with formation of abundant, hyaline extracellular matrix, and avoidance of hypertrophy being prime challenges. We recently demonstrated that parathyroid hormone-related protein (PTHrP) is a promising factor to direct chondrogenesis of MSCs towards an articular phenotype, since intermittent PTHrP application stimulated cartilage matrix production and reduced undesired hypertrophy. We here investigated the role of frequency, pulse duration, total exposure time, and underlying mechanisms in order to unlock the full potential of PTHrP actions. Human MSC subjected to in vitro chondrogenesis for six weeks were exposed to 2.5 nM PTHrP(1-34) pulses from days 7 to 42. Application frequency was increased from three times weekly (3 × 6 h/week) to daily maintaining either the duration of individual pulses (6 h/day) or total exposure time (18 h/week; 2.6 h/day). Daily PTHrP treatment significantly increased extracellular matrix deposition regardless of pulse duration and suppressed alkaline-phosphatase activity by 87%. High total exposure time significantly reduced cell proliferation at day 14. Pulse duration was critically important to significantly reduce IHH expression, but irrelevant for PTHrP-induced suppression of the hypertrophic markers MEF2C and IBSP. COL10A1, RUNX2, and MMP13 expression remained unaltered. Decreased IGFBP-2, -3, and -6 expression suggested modulated IGF-I availability in PTHrP groups, while drop of SOX9 protein levels during the PTHrP-pulse may delay chondroblast formation and hypertrophy. Overall, the significantly optimized timing of PTHrP-pulses demonstrated a vast potential to enhance chondrogenesis of MSC and suppress hypertrophy possibly via superior balancing of IGF- and SOX9-related mechanisms. J. Cell. Physiol. 231: 2673-2681, 2016. © 2016 Wiley Periodicals, Inc.