[show abstract][hide abstract] ABSTRACT: An important goal of stem cell research in orthopaedics is to develop clinically relevant techniques that could be applied to heal cartilage or joint pathology. Stem cell treatment in orthopaedics for joint pathology is promising since these cells have the ability to modulate different processes in the various tissues of the joint simultaneously. The non life-threatening nature of musculoskeletal system disorders makes safety of stem cell therapy a necessary prerequisite.
To systematically review the literature and provide an overview of reported adverse events of intra-articular treatment with culture-expanded stem cells in humans.
A systematic literature search was performed in Pubmed, EMBASE, Web of Science and CINAHL in February 2013. Adverse events were reported into three categories: local/systemic, serious adverse event or adverse event (SAE/AE), related/unrelated.
3039 potentially eligible articles were identified of which eventually 8 fulfilled our inclusion criteria. In total, 844 procedures with a mean follow-up of 21 months were analysed. Autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) were used for cartilage repair and osteoarthritis treatment in all included studies. Four serious adverse events (SAE) were reported by the authors. One infection following bone marrow aspiration (BMA) was reported as probably related and resolved with antibiotics. One pulmonary embolism occurred 2 weeks after bone marrow aspiration and was reported as possibly related. Two tumours, both not at the site of injection, were reported as unrelated. Twenty-two other cases of possible procedure-related and seven of possible stem cell-product related adverse events were documented. The main adverse events related to the procedure were increased pain/swelling and dehydration after BMA. Increased pain and swelling was the only adverse event reported as related to the stem cell-product.
Based on current literature review we conclude that application of cultured stem cells in joints appears to be safe. We believe that with continuous caution for potential side effects, it is reasonable to continue with the development of articular stem cell therapies.
Osteoarthritis and Cartilage 07/2013; · 4.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Previous studies have shown accumulation and an enhanced proinflammatory profile of macrophages in adipose tissue of obese mice, indicating the presence of an interaction between adipocytes and macrophages in this tissue. However, the consequences of this interaction in humans are yet incompletely understood. In this study, we explored the modulating effects of adipocytes on the phenotype of macrophages in humans and studied the possible molecular pathways involved. Adipocyte-conditioned media (ACM) treatment of macrophages for 48 h strongly reduced the LPS-induced IL-12p40 secretion by macrophages, whereas the production of TNF-α and other cytokines remained largely unaffected. This effect was independent of the source of adipocytes. Interestingly, the level of inhibition correlated directly with body mass index (BMI) of the adipocyte donor. Because adipocytes release many different cytokines, adipokines, and lipids, we have separated the protein and lipid fractions of ACM, to obtain insight into the molecular nature of the soluble mediators underlying the observed effect. These experiments revealed that the inhibitory effect resided predominantly in the lipid fraction. Further studies revealed that PGE2 and linoleic and oleic acid were potent inhibitors of IL-12p40 secretion. Interestingly, concentrations of these ACM-derived lipids increased with increase in BMI of the adipocyte donor, suggesting that they could mediate the BMI-dependent effects of ACM. To our knowledge, these results provide first evidence that obesity-related changes in adipose tissue macrophage phenotype could be mediated by adipocyte-derived lipids in humans. Intriguingly, these changes appear to be different from those in murine obesity.
The Journal of Immunology 07/2013; · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fibrates are used as lipid-lowering drugs to prevent cardiovascular pathology. Fibrates are ligands of peroxisome proliferator-activated receptor α (PPARα). Besides altering lipid metabolism, PPARα ligands exert anti-inflammatory effects on various cell types. In this study, we hypothesized that PPARα agonists exert beneficial effects on osteoarthritis (OA) and rheumatoid arthritis (RA) by their local anti-inflammatory effects, but also by their systemic influences. A systematic literature search of Medline and EMBASE databases was performed up to August 2011. The main search items were osteoarthritis, rheumatoid arthritis, peroxisome proliferator-activated receptor alpha and fibrates. Inclusion criteria were in vivo or in vitro studies regarding humans or animals in which the effects of PPARα ligands were studied. Six in vivo human studies, four in vivo animal studies and seven in vitro studies were included. The in vivo human studies showed all beneficial clinical effects of PPARα ligands, but studies were small and only four were randomized. Ligands for PPARα significantly reduced pain, swelling of the joints and decreased systemic inflammatory markers. In vitro and in vivo animal studies indicate that PPARα agonists inhibit bone resorption, and reduce inflammatory and destructive responses in cartilage and synovium. PPARα agonists such as fibrates should be considered as potential therapeutic strategy for RA. There is no clinical evidence for their use in OA, although in vitro studies indicate that PPARα agonists demonstrate different joint-protective effects locally, and systemic effects on inflammation, serum lipid levels and vascular pathology. Animal studies should be performed and after confirmation of the protective effects of PPARα, large randomized controlled trials could investigate fibrates in OA and RA.
Therapeutic advances in musculoskeletal disease 02/2013; 5(1):33-44.
[show abstract][hide abstract] ABSTRACT: Evidence is accumulating that synovial tissue plays an active role in osteoarthritis (OA), however, exact understanding of its contribution is lacking. In order to further elucidate its role in the OA process, we aimed to identify the secretion pattern of soluble mediators by synovial tissue and to assess its ability to initiate cartilage degeneration.
Synovial tissue explants (STEs) obtained from donors without history of OA (n = 8) or from end stage OA patients (n = 16) were cultured alone or together with bovine cartilage explants in the absence or presence of IL-1α. The secretion of 48 soluble mediators was measured and the effect on glycosaminoglycan (GAG) release and matrix metalloproteinase (MMP) activity was determined.
Normal and OA STEs secreted comparable levels of almost all measured soluble mediators. However, in the presence of IL-1α these mediators were less secreted by OA than by normal STEs of which 15 differed significantly (p<0.01). No effect of normal or OA STEs on GAG release from the cartilage explants was observed, and no differences in MMP activity between OA and normal STEs were detected.
Unexpectedly, a comparable secretion profile of soluble mediators was found for OA and normal STEs while the reduced responsiveness of OA STEs to an inflammatory trigger indicates a different state of this tissue in OA patients. The effects could be the result of prolonged exposure to an inflammatory environment in OA development. Further understanding of the pro-inflammatory and inflammation resolving mechanisms during disease progression in synovial tissue may provide valuable targets for therapy in the future.
PLoS ONE 01/2013; 8(5):e62634. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adipose tissue-derived mesenchymal stem cells (ASC) are of great interest as a cellular therapeutic agent for regenerative and immunomodulatory purposes. The function of ASC adapts to environmental conditions, such as oxygen tension. Oxygen levels within tissues are typically much lower than under standard culture conditions and ASC used for therapy therefore encounter a change from normoxic to hypoxic conditions. The effect of hypoxia on the regenerative potential of ASC has been investigated in a number of studies. The effect of hypoxia on the immunomodulatory function of ASC, however, remains to be determined. In the present study the effect of hypoxic (1% oxygen) culture conditions on human ASC was examined. ASC showed no signs of toxicity under low oxygen levels and no major immunophenotypical changes were observed, apart from a down regulation of the marker CD105. Oxygen tension had no effect on the proliferation of ASC and colony forming unit efficiency remained the same under 1 and 20% oxygen. Under both oxygen levels ASC were capable of strong upregulation of the immunomodulatory molecules indoleamine 2,3-dioxygenase (IDO) and programed death ligand-1 upon stimulation with IFN-γ and TNF-α, and, in addition, IDO activity as measured by the accumulation of l-kynurenine was not affected under hypoxia. The ability of ASC to inhibit anti-CD3/CD28 stimulated CD4(+) and CD8(+) T cell proliferation was not hampered by hypoxia. The results of the present study demonstrate that the immunosuppressive capacity of ASC is maintained under hypoxic conditions. These findings are important for the therapeutic use of ASC and may be applied for the in vitro generation of ASC with improved functionality for therapeutic use.
[show abstract][hide abstract] ABSTRACT: Infrapatellar fat pad (IPFP) might be involved in osteoarthritis (OA) by production of cytokines. It was hypothesised that production of cytokines is sensitive to environmental conditions.
To evaluate cytokine production by IPFP in response to interleukin (IL)1β and investigate the ability to modulate this response with an agonist for peroxisome proliferator activated receptor α (PPARα), which is also activated by lipid-lowering drugs such as fibrates.
Cytokine secretion of IPFP was analysed in the medium of explant cultures of 29 osteoarthritic patients. IPFP (five donors) and synovium (six donors) were cultured with IL-1β and PPARα agonist Wy14643. Gene expression of IL-1β, monocyte chemoattractant protein (MCP1), (IL-6, tumour necrosis factor (TNF)α, leptin, vascular endothelial growth factor (VEGF), IL-10, prostaglandin-endoperoxide synthase (PTGS)2 and release of TNFα, MCP1 and prostaglandin E(2) were compared with unstimulated IPFP and synovium explants.
IPFP released large amounts of inflammatory cytokines, adipokines and growth factors. IL-1β increased gene expression of PTGS2, TNFα, IL-1β, IL-6 and VEGF and increased TNFα release in IPFP. MCP1, leptin, IL-10 gene expression and MCP1, leptin and PGE(2) release did not increase significantly. Synovium responded to IL-1β similarly to IPFP, except for VEGF gene expression. Wy14643 decreased gene expression of PTGS2, IL-1β, TNFα, MCP1, VEGF and leptin in IPFP explants and IL-1β, TNFα, IL-6, IL-10 and VEGF in synovium that responded to IL-1β.
IPFP is an active tissue within the joint. IPFP cytokine production is increased by IL-1β and decreased by a PPARα agonist. The effects were similar to effects seen in synovium. Fibrates may represent a potential disease-modifying drug for OA by modulating inflammatory properties of IPFP and synovium.
Annals of the rheumatic diseases 02/2012; 71(6):1012-8. · 8.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adipose tissue is known to release inflammatory cytokines and growth factors. In this exploratory study, the authors examined whether the infrapatellar fat pad (IPFP) closely located to cartilage in the knee joint can affect cartilage metabolism. In addition, the authors analysed whether the macrophage types present in IPFP could explain the effect on cartilage.
IPFP explants obtained during total knee replacement of 29 patients with osteoarthritis (OA) were used to make fat-conditioned medium (FCM). Explants of bovine cartilage were cultured with or without FCM. Nitric oxide (NO) and glycosaminoglycan release and gene expression of matrix-degrading enzymes in cartilage were analysed. To stimulate catabolic processes in the cartilage, the authors added interleukin 1β, and the effect of six FCMs was evaluated. The presence of different types of macrophages (CD68+, CD86+ and CD206+) in OA IPFPs was compared with subcutaneous adipose tissue samples and IPFP samples from patients with an anterior cruciate ligament rupture.
FCM alone reduced NO and glycosaminoglycan release and matrix metalloproteinase (MMP)1 gene expression by the cartilage. Moreover, when catabolic conditions were enhanced with interleukin 1β, FCM inhibited NO production as well as MMP1 and MMP3 gene expression and increased collagen type II gene expression. Significantly more CD206+ cells were present in OA IPFP samples than in subcutaneous fat or anterior cruciate ligament IPFP samples.
In contrast to the authors' expectations, medium conditioned by end-stage OA IPFP inhibited catabolic processes in cartilage. CD206+ cells present in the IPFPs used for making the FCM might have contributed to the inhibition of catabolic processes in the cartilage.
Annals of the rheumatic diseases 02/2012; 71(2):288-94. · 8.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background: In diseased joints, the catabolic environment results in progressive joint damage. Mesenchymal stem cells (MSCs) can have immunomodulatory effects by secreting anti-inflammatory factors. To exert these effects, MSCs need to be triggered by pro-inflammatory cytokines. To explore the potential of MSCs as a treatment for diseased joints, we studied the effect of synovial fluid (SF) from donors with different joint diseases and donors without joint pathology on the immunomodulatory capacities of human MSCs in vitro. We hypothesized that SF of diseased joints influences the immunomodulatory effects of MSCs. Materials and Methods: MSCs were cultured in medium with SF of six osteoarthritis (OA) or six rheumatoid arthritis (RA) donors and three donors without joint pathology were used as control. Gene expressions of IL-6, HGF, TNFa, TGFb1, and indoleamine 2,3-dioxygenase (IDO) were analyzed. l-kynurenine concentration in conditioned medium (CM) by MSCs with SF was determined as a measure of IDO activity by MSCs. Furthermore, the effect of CM with SF on proliferation of activated lymphocytes was analyzed. Results: Addition of SF significantly up-regulated the mRNA expression of IL-6 and IDO in MSCs. SF(OA) induced significantly higher expression of IDO than SF(control), although no difference in IDO activity of the MSCs could be shown with a l-kynurenine assay. Medium conditioned by MSCs with SF(OA or RA) suppressed activated lymphocyte proliferation in vitro more than medium conditioned by MSCs without SF or with SF(control). Discussion: SF can influence the expression of genes involved in immunomodulation by MSCs and the effect on lymphocyte proliferation. We found indications for disease-specific differences between SFs but the variation between donors, even within one disease group was high. These data warrant further research to examine the potential application of MSC therapy in arthritic joints.
[show abstract][hide abstract] ABSTRACT: The increasing average age in developed societies is paralleled by an increase in the prevalence of many age-related diseases such as osteoarthritis (OA), which is characterized by deformation of the joint due to cartilage damage and increased turnover of subchondral bone. Consequently, deficiency in DNA repair, often associated with premature aging, may lead to increased pathology of these two tissues. To examine this possibility, we analyzed the bone and cartilage phenotype of male and female knee joints derived from 52- to 104-week-old WT C57Bl/6 and trichothiodystrophy (TTD) mice, who carry a defect in the nucleotide excision repair pathway and display many features of premature aging. Using micro-CT, we found bone loss in all groups of 104-week-old compared to 52-week-old mice. Cartilage damage was mild to moderate in all mice. Surprisingly, female TTD mice had less cartilage damage, proteoglycan depletion, and osteophytosis compared to WT controls. OA severity in males did not significantly differ between genotypes, although TTD males had less osteophytosis. These results indicate that in premature aging TTD mice age-related changes in cartilage were not more severe compared to WT mice, in striking contrast with bone and many other tissues. This segmental aging character may be explained by a difference in vasculature and thereby oxygen load in cartilage and bone. Alternatively, a difference in impact of an anti-aging response, previously found to be triggered by accumulation of DNA damage, might help explain why female mice were protected from cartilage damage. These findings underline the exceptional segmental nature of progeroid conditions and provide an explanation for pro- and anti-aging features occurring in the same individual.
[show abstract][hide abstract] ABSTRACT: Although several treatments for cartilage repair have been developed and used in clinical practice the last 20 years, little is known about the mechanisms that are involved in the formation of repair tissue after these treatments. Often, these treatments result in the formation of fibrocartilaginous tissue rather than normal articular cartilage. Because the repair tissue is inferior to articular cartilage in terms of mechanical properties and zonal organization of the extracellular matrix, complaints of the patient may return. The biological and functional outcome of these treatments should thus be improved. For this purpose, an in vitro model allowing investigation of the involved repair mechanisms can be of great value. We present the development of such a model. We used bovine osteochondral biopsies and created a system in which cartilage defects of different depths can be studied. First, our biopsy model was characterized extensively: we studied the viability by means of lactate dehydrogenase (LDH) excretion over time and we investigated expression of cartilage-related genes in osteochondral biopsies and compared it with conventional cartilage-only explants. After 28 days of culture, LDH was detected at low levels and mRNA could be retrieved. The expression of cartilage-related genes decreased over time. This was more evident in cartilage-only explants, indicating that the biopsy model provided a more stable environment. We also characterized the subchondral bone: osteoclasts and osteoblasts were active after 28 days of culture, which was indicated by tartrate acid phosphatase staining and alkaline phosphatase measurements, respectively, and matrix deposition during culture was visualized using calcein labeling. Second, the applicability of the model was further studied by testing two distinct settings: (1) implantation of chondrocytes in defects of different depths; (2) two different seeding strategies of chondrocytes. Differences were observed in terms of volume and integration of newly formed tissue in both settings, suggesting that our model can be used to model distinct conditions or even to mimic clinical treatments. After extensive characterization and testing of our model, we present a representative and reproducible in vitro model that can be used to evaluate new cartilage repair treatments and study mechanisms in a controlled and standardized environment.
Tissue Engineering Part C Methods 08/2011; 18(1):45-53. · 4.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Platelet-rich plasma (PRP) has recently been postulated as a treatment for osteoarthritis (OA). Although anabolic effects of PRP on chondrocytes are well documented, no reports are known addressing effects on cartilage degeneration. Since OA is characterized by a catabolic and inflammatory joint environment, the authors investigated whether PRP was able to counteract the effects of such an environment on human osteoarthritic chondrocytes.
Platelet-rich plasma inhibits inflammatory effects of interleukin-1 (IL-1) beta on human osteoarthritic chondrocytes.
Controlled laboratory study.
Human osteoarthritic chondrocytes were cultured in the presence of IL-1 beta to mimic an osteoarthritic environment. Medium was supplemented with 0%, 1%, or 10% PRP releasate (PRPr, the active releasate of PRP). After 48 hours, gene expression of collagen type II alpha 1 (COL2A1), aggrecan (ACAN), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, matrix metalloproteinase (MMP)13, and prostaglandin-endoperoxide synthase (PTGS)2 was analyzed. Additionally, glycosaminoglycan (GAG) content, nitric oxide (NO) production, and nuclear factor kappa B (NFκB) activation were studied.
Platelet-rich plasma releasate diminished IL-1 beta-induced inhibition of COL2A1 and ACAN gene expression. The PRPr also reduced IL-1 beta-induced increase of ADAMTS4 and PTGS2 gene expression. ADAMTS5 gene expression and GAG content were not influenced by IL-1 beta or additional PRPr. Matrix metalloproteinase 13 gene expression and NO production were upregulated by IL-1 beta but not affected by added PRPr. Finally, PRPr reduced IL-1 beta-induced NFκB activation to control levels containing no IL-1 beta.
Platelet-rich plasma releasate diminished multiple inflammatory IL-1 beta-mediated effects on human osteoarthritic chondrocytes, including inhibition of NFκB activation.
Platelet-rich plasma releasate counteracts effects of an inflammatory environment on genes regulating matrix degradation and formation in human chondrocytes. Platelet-rich plasma releasate decreases NFκB activation, a major pathway involved in the pathogenesis of OA. These results encourage further study of PRP as a treatment for OA.
The American journal of sports medicine 08/2011; 39(11):2362-70. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to create and characterize a tissue-engineered mucosal equivalent (TEM) that closely resembles native mucosa. TEM consists of human primary keratinocytes and fibroblasts isolated from biopsies taken from healthy donors and seeded onto a de-epidermized dermis and cultured for 14 days at the air/liquid interface. The structure of TEM was examined and compared with native nonkeratinizing oral mucosa (NNOM). The various components of the newly formed epidermal layer, basement membrane and underlying connective tissue were analyzed using immunohistochemistry. The mucosal substitute presented in this study showed a mature stratified squamous epithelium that was similar to that of native oral mucosa, as demonstrated by K19, desmoglein-3 and involucrin staining. In addition, the expression of basement membrane components collagen type IV, laminin-5 and integrin α6 and β4 in TEM proved to be consistent with native oral mucosa. The expression of PAS, Ki67, K10 and K13, however, appeared to be different in TEM compared to NNOM. Nevertheless, the similarities with native oral mucosa makes TEM a promising tool for studying the biology of mucosal pathologies such as oral mucositis or fibrosis as well as the development of new therapies.
[show abstract][hide abstract] ABSTRACT: Although both cartilage and synovium are affected in osteoarthritis (OA), no in vitro coculture models of human OA tissue have been described. The aim of this study was to develop an in vitro model that includes both the synovium and cartilage of patients with knee OA.
Explants of human OA cartilage and synovium were cultured alone or in coculture for 21 days. Histologic evaluation and analyses of lactate dehydrogenase release, matrix metalloproteinase (MMP) activity, content, release, and synthesis of glycosaminoglycan (GAG), and cytokine production were used to evaluate synovial tissue functionality and its effect on cartilage metabolism. To assess the possibility of intervention in the model system, the effect of triamcinolone was studied.
Throughout the entire culture period, OA synovial tissue remained viable and produced cytokines. Monocultures of synovial and cartilage explants produced different cytokine subsets, with the subsets found in coculture being most similar to those previously described in OA synovial fluid. MMP activity was detectable only in the synovial explant monoculture and in coculture. Cocultures showed a reduction in final GAG content (P < 0.02), attributable to an inhibition of GAG production (P < 0.001) rather than an increase in GAG release. Addition of triamcinolone inhibited cytokine production and MMP activity in coculture and synovial tissue monoculture and counteracted the inhibition of GAG production induced by coculture. In cartilage monoculture, however, triamcinolone reduced GAG production.
OA synovium affects cartilage metabolism by reducting GAG production. Triamcinolone can relieve this effect of synovial tissue, while being inhibitory when added to cartilage monoculture. These results clearly indicate the importance of tissue coculture as a promising tool for studying OA pathophysiology and for development of possible interventions.
[show abstract][hide abstract] ABSTRACT: In osteoarthritis (OA), changes occur in both cartilage and subchondral bone. The subchondral bone plate facilitates normal cross-talk between articular cartilage and trabecular subchondral bone, and adaptive changes in the plate due to OA may therefore disturb cross-talk homeostasis. To investigate these changes over time, we examined the cartilage-subchondral bone interface using a combined approach of histologic analysis and in vivo microfocal computed tomography.
Sixteen-week-old male C57BL/6 mice (n=32) received intraarticular injections of collagenase in 1 joint to induce instability-related OA and received saline injections in the contralateral knee joint (control joint). At 2, 4, 6, 10, and 14 weeks after injection, changes in the tibial subchondral bone plate and subchondral trabeculae were analyzed.
Two weeks after injection, collagenase-injected joints had significantly more cartilage damage and osteophytosis than did control joints. Osteoclast activity directly underneath the subchondral bone plate was significantly elevated in collagenase-injected joints compared to control joints (mean±SEM osteoclast surface/bone surface 11.07±0.79% versus 7.60±0.81%), causing the plate to become thinner and creating a large increase in subchondral bone plate porosity (mean±SEM cumulative porosity volume 0.05±0.04×10(-3) mm3 in control joints versus 2.52±0.69×10(-3) mm3 in collagenase-injected joints). Four weeks after injection, the previously formed perforations disappeared, coinciding with a significant rise in osteoblast activity in the subchondral trabecular bone in collagenase-injected joints compared to control joints (mean ± SEM bone formation rate/bone surface 0.62±0.13 μm3/μm2 per day versus 0.30±0.03 μm3/μm2 per day).
The current study is the first to provide quantitative longitudinal data on the dynamic changes in the subchondral bone plate after OA induction. The development of plate perforations may enhance mutual interaction between subchondral trabeculae, bone marrow cells, and the articular cartilage in OA.
[show abstract][hide abstract] ABSTRACT: Adipose regeneration strategies have been hampered by the inability to supply an adequate vascular supply following implantation. Vascularization in vitro, also called prevascularization, is a promising method that could promote the vascularization of engineered adipose tissue constructs upon implantation. In this study we compared the ability of prevascularized-to-non-prevascularized fibrin-based human adipose tissue to promote vascularization. Human adipose tissue-derived stromal cells (ASCs) and different mixtures (1:1, 1:2 and 1:5) of ASCs with human umbilical vein endothelial cells (HUVECs) were cultured in fibrin at two different densities (1.0 × 10(6) and 10 × 10(6) cells/ml) for 7 days. Histological analysis revealed that prevascular structures formed in 1:5 ASC/HUVEC fibrin-based constructs seeded with a total of 10 × 10(6) cells/ml. These constructs and ASC-only constructs were implanted subcutaneously in athymic mice for 7 days and generated lipid-containing grafts. The numbers and densities of blood vessels within the ASC/HUVEC constructs were similar to those of ASC-only constructs. Furthermore, immunostaining studies demonstrated human-derived vasculature within a few of the ASC/HUVEC and ASC-only constructs. A subset of this human-derived vasculature contained erythrocytes, indicating integration with the host vasculature. In conclusion, our study indicated no difference in the rate of vascularization of prevascularized ASC/HUVEC and non-prevascularized ASC-only fibrin-based constructs, suggesting that prevascularization of these fibrin-based constructs does not promote vascularization. Our results further indicated that not only endothelial cells, but also ASCs may contribute to the formation of vascular lumina upon implantation. This finding is interesting, since it demonstrates the possibility of vascularized adipose tissue engineering from a single cell source.
Journal of Tissue Engineering and Regenerative Medicine 02/2011; 6(3):169-78. · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.
Tissue Engineering Part A 01/2011; 17(7-8):1157-67. · 4.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway.
MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture.
After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin.
The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of β-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Articular cartilage has very limited intrinsic regenerative capacity, making cell-based therapy a tempting approach for cartilage repair. Cell tracking can be a major step towards unraveling and improving the repair process of these therapies. We studied superparamagnetic iron oxides (SPIO) for labeling human bone marrow-derived mesenchymal stem cells (hBMSCs) regarding effectivity, cell viability, long term metabolic cell activity, chondrogenic differentiation and hBMSC secretion profile. We additionally examined the capacity of synovial cells to endocytose SPIO from dead, labeled cells, together with the use of magnetic resonance imaging (MRI) for intra-articular visualization and quantification of SPIO labeled cells. METHODOLOGY/PRINICIPAL FINDINGS: Efficacy and various safety aspects of SPIO cell labeling were determined using appropriate assays. Synovial SPIO re-uptake was investigated in vitro by co-labeling cells with SPIO and green fluorescent protein (GFP). MRI experiments were performed on a clinical 3.0T MRI scanner. Two cell-based cartilage repair techniques were mimicked for evaluating MRI traceability of labeled cells: intra-articular cell injection and cell implantation in cartilage defects. Cells were applied ex vivo or in vitro in an intra-articular environment and immediately scanned. SPIO labeling was effective and did not impair any of the studied safety aspects, including hBMSC secretion profile. SPIO from dead, labeled cells could be taken up by synovial cells. Both injected and implanted SPIO-labeled cells could accurately be visualized by MRI in a clinically relevant sized joint model using clinically applied cell doses. Finally, we quantified the amount of labeled cells seeded in cartilage defects using MR-based relaxometry. CONCLUSIONS: SPIO labeling appears to be safe without influencing cell behavior. SPIO labeled cells can be visualized in an intra-articular environment and quantified when seeded in cartilage defects.
PLoS ONE 01/2011; 6(2):e17001. · 3.73 Impact Factor