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Background: Cartilage defects result in joint inflammation. The presence of proinflammatory factors has been described to negatively affect cartilage formation. Purpose: To evaluate the effect and timing of administration of triamcinolone acetonide (TAA), an anti-inflammatory drug, on cartilage repair using a mouse model. Study Design: Controlled laboratory study. Methods: A full-thickness cartilage defect was created in the trochlear groove of 10-week-old male DBA/1 mice (N = 80). Mice received an intra-articular injection of TAA or saline on day 1 or 7 after induction of the defect. Mice were euthanized on days 10 and 28 for histological evaluation of cartilage defect repair, synovial inflammation, and synovial membrane thickness. Results: Mice injected with TAA had significantly less synovial inflammation at day 10 than saline-injected mice independent of the time of administration. At day 28, the levels of synovitis dropped toward healthy levels; nevertheless, the synovial membrane was thinner in TAA- than in saline-injected mice, reaching statistical significance in animals injected on day 1 (70.1 ± 31.9 µm vs 111.9 ± 30.9 µm, respectively; P = .01) but not in animals injected on day 7 (68.2 ± 21.86 µm vs 90.2 ± 21.29 µm, respectively; P = .26). A thinner synovial membrane was moderately associated with less filling of the defect after 10 and 28 days ( r = 0.42, P = .02; r = 0.47, P = .01, respectively). Whereas 10 days after surgery there was no difference in the area of the defect filled and the cell density in the defect area between saline- and TAA-injected knees, filling of the defect at day 28 was lower in TAA- than in saline-injected knees for both injection time points (day 1 injection, P = .04; day 7 injection, P = .01). Moreover, there was less collagen type 2 staining in the filled defect area in TAA- than in saline-injected knees after 28 days, reaching statistical significance in day 1–injected knees (2.6% vs 18.5%, respectively; P = .01) but not in day 7–injected knees (7.4% vs 15.8%, respectively; P = .27). Conclusion: Intra-articular injection of TAA reduced synovial inflammation but negatively affected cartilage repair. This implies that inhibition of inflammation may inhibit cartilage repair or that TAA has a direct negative effect on cartilage formation. Clinical Relevance: Our findings show that TAA can inhibit cartilage defect repair. Therefore, we suggest not using TAA to reduce inflammation in a cartilage repair setting.

Macrophages play an important role in the development and progression of osteoarthritis (OA). The aim of this study was to identify macrophage phenotypes in synovium and monocyte subsets in peripheral blood in C57BL/6 mice by destabilizing the medial meniscus (DMM), and the association of macrophage subsets with OA features. DMM, sham, and non-operated knees were histologically assessed between 1 and 56 days for macrophage polarization states by immunohistochemistry (IHC), cartilage damage, synovial thickening, and osteophytes (n=9 per timepoint). Naive knees (n=6) were used as controls. Monocyte and polarized synovial macrophage subsets were evaluated by flow cytometry. CD64 and CD206 levels on IHC were higher at early timepoints in DMM and sham knees compared to naive knees. iNOS labeling intensity was higher in DMM and sham knees than in naive knees from d3 onwards. CD163 expression was unaltered at all timepoints. Even though macrophage polarization profiles were similar in DMM and sham knees, only in DMM knees the presence of iNOS and CD206 associated with synovial thickness, and CD163 staining inversely correlated with osteophyte presence. At day 14, monocyte subset distribution was different in peripheral blood of DMM compared to sham mice. In conclusion, monocyte subsets in blood and synovial macrophage phenotypes vary after joint surgery. High levels of iNOS+ , CD163+ , and CD206+ cells are found in both destabilized and sham-operated knees, and co-existence with joint instability may be a requirement to initiate and exacerbate OA progression. This article is protected by copyright. All rights reserved.

Objective The potential chondroprotective effect of celecoxib, a nonsteroidal anti-inflammatory drug and selective cyclooxygenase-2 inhibitor used to reduce pain and inflammation in knee osteoarthritis patients, is disputed. This study aimed at investigating the chondroprotective effects of celecoxib on (1) human articular cartilage explants and (2) in an in vivo osteoarthritis rat model. Design Articular cartilage explants from 16 osteoarthritis patients were cultured for 24 hours with celecoxib or vehicle. Secreted prostaglandins (prostaglandin E2, prostaglandin F2α, prostaglandin D2) and thromboxane B2 (TXB2) concentrations were determined in medium by ELISA, and protein regulation was measured with label-free proteomics. Cartilage samples from 7 of these patients were analyzed for gene expression using real-time quantitative polymerase chain reaction. To investigate the chondroprotective effect of celecoxib in vivo, 14 rats received an intra-articular injection of celecoxib or 0.9% NaCl after osteoarthritis induction by anterior cruciate ligament transection and partial medial meniscectomy (ACLT/pMMx model). Histopathological scoring was used to evaluate osteoarthritis severity 12 weeks after injection. Results Secretion of prostaglandins, target of Nesh-SH3 (ABI3BP), and osteonectin proteins decreased, whereas tissue inhibitor of metalloproteinase 2 (TIMP-2) increased significantly after celecoxib treatment in the human ( ex vivo) explant culture. Gene expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS4/5) and metalloproteinase 13 (MMP13) was significantly reduced after celecoxib treatment in human cartilage explants. Cartilage degeneration was reduced significantly in an in vivo osteoarthritis knee rat model. Conclusions Our data demonstrated that celecoxib acts chondroprotective on cartilage ex vivo and a single intra-articular bolus injection has a chondroprotective effect in vivo.

Objective In osteoarthritis, chondrocytes tend to acquire a hypertrophic phenotype, which contributes to the modification of the extracellular matrix, resulting in permanent cartilage changes. In mouse chondrocytes, pro-inflammatory macrophages and pro-inflammatory cytokines have been shown to stimulate hypertrophy via the activation of the nuclear factor kappa B (NF-κB) pathway. Whether or not this also occurs in human chondrocytes remains unclear. We therefore aimed to investigate whether hypertrophy-like responses in human cartilage are driven mainly by intrinsic inflammatory signaling or shaped by specific macrophage populations. Design Human articular chondrocytes were cultured with pro-inflammatory cytokines or medium conditioned by defined macrophage subsets. Furthermore, the effect of inhibition of NF-κB-dependent gene expression was evaluated using the NF-κB inhibitor SC-514. Hypertrophy was assessed by measuring the transcription level of alkaline phosphatase ( ALPL), type X collagen ( COL10A1), Indian hedgehog ( IHH), and runt-related transcription factor 2 ( RUNX2). Results The expression of hypertrophic genes was not promoted in human chondrocytes by pro-inflammatory cytokines neither pro-inflammatory M(IFNγ + TNFα) macrophages. Inhibition of the NF-κB-dependent gene expression did not affect human articular chondrocyte hypertrophy. However, tissue repair M(IL4) macrophages induced hypertrophy by promoting the expression of COL10A1, RUNX2, and IHH. Conclusion Intrinsic inflammatory signaling activation is not involved in the hypertrophic shift observed in human articular chondrocytes cultured in vitro. However, tissue repair macrophages may contribute to the onset of this detrimental phenotype in human osteoarthritic cartilage, given the effect observed in our experimental models.

Mesenchymal stem cells (MSC) are promising candidates for use as a biological therapeutic. Since locally injected MSC disappear within a few weeks, we hypothesize that efficacy of MSC can be enhanced by prolonging their presence. Previously, encapsulation in alginate was suggested as a suitable approach for this purpose. We found no differences between the two alginate types, alginate high in mannuronic acid (High M) and alginate high in guluronic acid (High G), regarding MSC viability, MSC immunomodulatory capability, or retention of capsule integrity after subcutaneous implantation in immune competent rats. High G proved to be more suitable for production of injectable beads. Firefly luciferase-expressing rat MSC were used to track MSC viability. Encapsulation in high G alginate prolonged the presence of metabolically active allogenic MSC in immune competent rats with monoiodoacetate-induced osteoarthritis for at least 8 weeks. Encapsulation of human MSC for local treatment by intra-articular injection did not significantly influence the effect on pain, synovial inflammation, or cartilage damage in this disease model. MSC encapsulation in alginate allows for an injectable approach which prolongs the presence of viable cells subcutaneously or in an osteoarthritic joint. Further fine tuning of alginate formulation and effective dosage for might be required in order to improve therapeutic efficacy depending on the target disease.

Knee osteoarthritis (OA) is a condition mainly characterized by cartilage degradation. Currently, no effective treatment exists to slow down the progression of OA-related cartilage damage. Selective COX-2 inhibitors may, next to their pain killing properties, act chondroprotective in vivo. To determine whether the route of administration is important for the efficacy of the chondroprotective properties of selective COX-2 inhibitors, a systematic review was performed according to the PRISMA guidelines. Studies investigating OA-related cartilage damage of selective COX-2 inhibitors in vivo were included. Nine of the fourteen preclinical studies demonstrated chondroprotective effects of selective COX-2 inhibitors using systemic administration. Five clinical studies were included and, although in general non-randomized, failed to demonstrate chondroprotective actions of oral selective COX-2 inhibitors. All of the four preclinical studies using bolus intra-articular injections demonstrated chondroprotective actions, while one of the three preclinical studies using a slow release system demonstrated chondroprotective actions. Despite the limited evidence in clinical studies that have used the oral administration route, there seems to be a preclinical basis for considering selective COX-2 inhibitors as disease modifying osteoarthritis drugs when used intra-articularly. Intra-articularly injected selective COX-2 inhibitors may hold the potential to provide chondroprotective effects in vivo in clinical studies.

Objective To investigate GAG-ECM (glycosaminoglycan–extracellular matrix) interactions in different cartilage types. To achieve this, we first aimed to determine protocols for consistent calculation of GAG content between cartilage types. Design Auricular cartilage containing both collagen and elastin was used to determine the effect of lyophilization on GAG depletion activity. Bovine articular, auricular, meniscal, and nasal cartilage plugs were treated using different reagents to selectively remove GAGs. Sulfated glycosaminoglycan (sGAG) remaining in the sample after treatment were measured, and sGAG loss was compared between cartilage types. Results The results indicate that dry weight of cartilage should be measured prior to cartilage treatment in order to provide a more accurate reference for normalization. Articular, meniscal, and nasal cartilage lost significant amounts of sGAG for all reagents used. However, only hyaluronidase was able to remove significant amount of sGAG from auricular cartilage. Furthermore, hyaluronidase was able to remove over 99% of sGAG from all cartilage types except auricular cartilage where it only removed around 76% of sGAG. The results indicate GAG-specific ECM binding for different cartilage types and locations. Conclusions In conclusion, lyophilization can be performed to determine native dry weight for normalization without affecting the degree of GAG treatment. To our knowledge, this is the first study to compare GAG-ECM interactions of different cartilage types using different GAG extraction methods. Degree of GAG depletion not only varied with cartilage type but also the same type from different anatomic locations. This suggests specific structure-function roles for GAG populations found in the tissues.

Articular cartilage is frequently injured by trauma or osteoarthritis, with limited and inadequate treatment options. We investigated a new strategy based on hydrogel-mediated delivery of a locked nucleic acid microRNA inhibitor targeting miR-221 (antimiR-221) to guide in situ cartilage repair by endogenous cells. First, we showed that transfection of antimiR-221 into human bone marrow-derived mesenchymal stromal cells (hMSCs) blocked miR-221 expression and enhanced chondrogenesis in vitro. Next, we loaded a fibrin/hyaluronan (FB/HA) hydrogel with antimiR-221 in combination or not with lipofectamine carrier. FB/HA strongly retained functional antimiR-221 over 14 days of in vitro culture, and provided a supportive environment for cell transfection, as validated by flow cytometry and qRT-PCR analysis. Seeding of hMSCs on the surface of antimiR-221 loaded FB/HA led to invasion of the hydrogel and miR-221 knockdown in situ within 7 days. Overall, the use of lipofectamine enhanced the potency of the system, with increased antimiR-221 retention and miR-221 silencing in infiltrating cells. Finally, FB/HA hydrogels were used to fill defects in osteochondral biopsies that were implanted subcutaneously in mice. FB/HA loaded with antimiR-221/lipofectamine significantly enhanced cartilage repair by endogenous cells, demonstrating the feasibility of our approach and the need to achieve highly effective in situ transfection. Our study provides new evidence on the treatment of focal cartilage injuries using controlled biomaterial-mediated delivery of antimicroRNA for in situ guided regeneration.

Bone morphogenetic protein-2 (BMP-2) is a powerful osteoinductive protein; however, there is a need for the development of a safe and efficient BMP-2 release system for bone regeneration therapies. Recombinant extracellular matrix proteins are promising next generation biomaterials since the proteins are well-defined, reproducible and can be tailored for specific applications. In this study, we have developed a novel and versatile BMP-2 delivery system using microspheres from a recombinant protein based on human collagen I (RCP). In general, a two-phase release pattern was observed while the majority of BMP-2 was retained in the microspheres for at least two weeks. Among different parameters studied, the crosslinking and the size of the RCP micro spheres changed the in vitro BMP-2 release kinetics significantly. Increasing the chemical crosslinking (hexamethylene diisocyanide) degree decreased the amount of initial burst release (24 h) from 23% to 17%. Crosslinking by dehydrothermal treatment further decreased the burst release to 11%. Interestingly, the 50 and 72 mu m-sized spheres showed a significant decrease in the burst release compared to 207-mu m sized spheres. Very importantly, using a reporter cell line, the released BMP-2 was shown to be bioactive. SPR data showed that N-terminal sequence of BMP-2 was important for the binding and retention of BMP-2 and suggested the presence of a specific binding epitope on RCP (K-D: 1.2 nM). This study demonstrated that the presented RCP microspheres are promising versatile BMP-2 delivery vehicles.

Item does not contain fulltext Human bone marrow-derived mesenchymal stem cells (BMSCs) are clinically promising to repair damaged articular cartilage. This study investigated TWIST1, an important transcriptional regulator in mesenchymal lineages, in BMSC chondrogenesis. We hypothesized that downregulation of TWIST1 expression is required for in vitro chondrogenic differentiation. Indeed, significant downregulation of TWIST1 was observed in murine skeletal progenitor cells during limb development (N = 3 embryos), and during chondrogenic differentiation of culture-expanded human articular chondrocytes (N = 3 donors) and isolated adult human BMSCs (N = 7 donors), consistent with an inhibitory effect of TWIST1 expression on chondrogenic differentiation. Silencing of TWIST1 expression in BMSCs by siRNA, however, did not improve chondrogenic differentiation potential. Interestingly, additional investigation revealed that downregulation of TWIST1 in chondrogenic BMSCs is preceded by an initial upregulation. Similar upregulation is observed in non-chondrogenic BMSCs (N = 5 donors); however, non-chondrogenic cells fail to downregulate TWIST1 expression thereafter, preventing their chondrogenic differentiation. This study describes for the first time endogenous TWIST1 expression during in vitro chondrogenic differentiation of human BMSCs, demonstrating dynamic regulation of TWIST1 expression whereby upregulation and then downregulation of TWIST1 expression are required for chondrogenic differentiation of BMSCs. Elucidation of the molecular regulation of, and by, TWIST1 will provide targets for optimization of BMSC chondrogenic differentiation culture.

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation towards the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the anti-chondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs towards chondrogenic differentiation in the absence of TGF-β. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a pro-chondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. This article is protected by copyright. All rights reserved.

It is well-accepted that articular (ART) cartilage composition and tissue architecture are intimately related to mechanical properties. On the other hand, very little information about other cartilage tissues is available, such as elastin-rich auricular (AUR) cartilage. While thorough investigation of ART cartilage has enhanced osteoarthritis research, ear cartilage reconstruction and tissue engineering (TE) could benefit in a similar way from in-depth analysis of AUR cartilage properties. This study aims to explore the constituent-function relationships of AUR cartilage, and how elastin influences mechanical behavior. Stress-relaxation indentation and tensile tests were performed on bovine ART and AUR cartilage. Elastase incubation was performed to simultaneously deplete elastin and sulfated glycosaminoglycans (sGAG), while hyaluronidase incubation was used to deplete sGAG-only, in order to systematically investigate matrix components in material behavior. ART and AUR cartilages showed different viscoelastic behaviors, with AUR cartilage exhibiting a more elastic behavior. Higher equilibrium properties and limited viscous dissipation of strain energy were observed in AUR cartilage, while ART cartilage exhibited a rapid viscous response and high resistance to instantaneous loading. In conclusion, loss of sGAG had no effect on auricular mechanics in contrast to articular cartilage where GAG loss clearly correlated with mechanical properties. Auricular cartilage without elastin lost all compressive mechanical integrity, whereas in articular cartilage this was provided by collagen. This work shows for the first time the involvement of elastin in the mechanical behavior of ear cartilage. In future, this data can be used in AUR cartilage TE efforts to support reproduction of tissue-specific mechanical properties. (C) 2015 Elsevier Ltd. All rights reserved.

Recapitulation of endochondral ossification leads to a new concept of bone tissue engineering via a cartilage intermediate as an osteoinductive template. In this study, we aimed to investigate the influence of in vitro chondrogenic priming time for the creation of cartilage template on the in vivo endochondral bone formation both qualitatively and quantitatively. To this end, rat bone-marrow-derived mesenchymal stromal cells (MSCs) were seeded onto two scaffolds with distinguished features: a fibrous poly(lactic-co-glycolic acid)/poly(epsilon-caprolactone) electrospun scaffold (PLGA/PCL) and a porous hydroxyapatite/tricalcium phosphate composite (HA/TCP). The constructs were then chondrogenically differentiated for 2, 3 and 4 weeks in vitro, followed by subcutaneous implantation in vivo for up to 8 weeks. A longer chondrogenic priming time resulted in a significantly increased amount and homogeneous deposition of the cartilage matrix on both the PLGA/PCL and HA/TCP scaffolds in vitro. In vivo, all implanted constructs gave rise to endochondral bone formation, whereas the bone volume was not affected by the length of priming time. An unpolarized woven bone-like structure, with significant amounts of cartilage remaining, was generated in fibrous PLGA/PCL scaffolds, while porous HA/TCP scaffolds supported progressive lamellar-like bone formation with mature bone marrow development. These data suggest that, by utilizing a chondrogenically differentiated MSC-scaffold construct as cartilage template, 2 weeks of in vitro priming time is sufficient to generate a substantial amount of vascularized endochondral bone in vivo. The structure of the bone depends on the chemical and structural cues provided by the scaffold design. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Background: Osteoarthritis (OA) is a degenerative joint disease leading to pain and disability for which no curative treatment exists. A promising biological treatment for OA is intra-articular administration of platelet-rich plasma (PRP). PRP injections in OA joints can relieve pain, although the exact working mechanism is unclear. Purpose: To examine the effects of PRP releasate (PRPr) on pain, cartilage damage, and synovial inflammation in a mouse OA model. Study Design: Controlled laboratory study. Methods: OA was induced unilaterally in the knees of male mice (n = 36) by 2 intra-articular injections of collagenase at days –7 and –5. At day 0, pain was measured by registering weight distribution on the hindlimbs, after which mice were randomly divided into 2 groups. Mice received 3 intra-articular injections of PRP or saline in the affected knee. Seven mice per group were euthanized at day 5 for assessment of early synovial inflammation and cartilage damage. Pain in the remaining mice was registered for a total of 3 weeks. These mice were euthanized at day 21 for assessment of cartilage damage and synovial inflammation on histological evaluation. Antibodies against iNOS, CD163, and CD206 were used to identify different subtypes of macrophages in the synovial membrane. Results: Mice in the PRPr group increased the distribution of weight on the affected joint in 2 consecutive weeks after the start of the treatment ( P < .05), whereas mice in the saline group did not. At day 21, PRPr-injected knees had a thinner synovial membrane ( P < .05) and a trend toward less cartilage damage in the lateral joint compartment ( P = .053) than saline-injected knees. OA knees treated with saline showed less anti-inflammatory (CD206+ and CD163+) cells at day 5 than healthy knees, an observation that was not made in the PRPr-treated group. A higher level of pain at day 7 was associated with a thicker synovial membrane at day 21. The presence of CD206+ cells was negatively associated with synovial membrane thickness. Conclusion: In a murine OA model, multiple PRPr injections reduced pain and synovial thickness, possibly through modulation of macrophage subtypes. Clinical Relevance: PRPr injections in early OA or shortly after joint trauma can reduce pain and synovial inflammation and may inhibit OA development in patients.

Trauma and age-related cartilage disorders represent a major global cause of morbidity, resulting in chronic pain and disability in patients. A lack of effective therapies, together with a rapidly aging population, creates an impressive clinical and economic burden on healthcare systems. In this scenario, experimental therapies based on transplantation or in situ stimulation of skeletal Mesenchymal Stem/progenitor Cells (MSCs) have raised great interest for cartilage repair. Nevertheless, the challenge of guiding MSC differentiation and preventing cartilage hypertrophy and calcification still needs to be overcome. While research has mostly focused on the stimulation of cartilage anabolism using growth factors, several issues remain unresolved prompting the field to search for novel solutions. Recently, inhibition of anti-chondrogenic regulators has emerged as an intriguing opportunity. Anti-chondrogenic regulators include extracellular proteins as well as intracellular transcription factors and microRNAs that act as potent inhibitors of pro-chondrogenic signals. Suppression of these inhibitors can enhance MSC chondrogenesis and production of cartilage matrix. We here review the current knowledge concerning different types of anti-chondrogenic regulators. We aim to highlight novel therapeutic targets for cartilage repair and discuss suitable tools for suppressing their anti-chondrogenic functions. Further effort is needed to unveil the therapeutic perspectives of this approach and pave the way for effective treatment of cartilage injuries in patients. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptaLchondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 +/- 25 pm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 +/- 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 +/- 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses. (C) 2014 Elsevier Ltd. All rights reserved.

European Cells & Materials, 27, ISSN:1473-2262

Background: Mesenchymal stem cells (MSCs) are promising candidates as a cell-based therapy for osteoarthritis (OA), although current results are modest. Pre-treatment of MSCs before application might improve their therapeutic efficacy. Hypothesis: Pre-treatment of MSCs with inflammatory factors or hypoxia will improve their migration and adhesion capacities toward OA-affected tissues. Study Design: Controlled laboratory study. Methods: We used real-time polymerase chain reaction to determine the effects of different fetal calf serum (FCS) batches, platelet lysate (PL), hypoxia, inflammatory factors, factors secreted by OA tissues, and OA synovial fluid (SF) on the expression of 12 genes encoding chemokine or adhesion receptors. Migration of MSCs toward factors secreted by OA tissues was studied in vitro, and attachment of injected MSCs was evaluated in vivo in healthy and OA knees of male Wistar rats. Results: Different FCS batches, PL, or hypoxia did not influence the expression of the migration and adhesion receptor genes. Exposure to inflammatory factors altered the expression of CCR1, CCR4, CD44, PDGFRα, and PDGFRβ. MSCs migrated toward factors secreted by OA tissues in vitro. Neither pre-treatment with inflammatory factors nor the presence of OA influenced MSC migration in vitro or adhesion in vivo. Conclusion: Factors secreted by OA tissues increase MSC migration in vitro. In vivo, no difference in MSC adhesion was found between OA and healthy knees. Pre-treatment with inflammatory factors influenced the expression of migration and adhesion receptors of MSCs but not their migration in vitro or adhesion in vivo. Clinical Relevance: To improve the therapeutic capacity of intra-articular injection of MSCs, they need to remain intra-articular for a longer period of time. Pre-treatment of MSCs with hypoxia or inflammatory factors did not increase the migration or adhesion capacity of MSCs and will therefore not likely prolong their intra-articular longevity. Alternative approaches to prolong the intra-articular presence of MSCs should be developed to increase the therapeutic effect of MSCs in OA.

Controlled biomaterial-based corticosteroid release might circumvent multiple injections and the accompanying risks, such as hormone imbalance and muscle weakness, in osteoarthritic (OA) patients. For this purpose, microspheres were prepared from an amino acid-based polyester amide (PEA) platform and loaded with triamcinolone acetonide (TAA). TAA loaded microspheres were shown to release TAA for over 60days in PBS. Furthermore, the bioactivity lasted at least 28days, demonstrated by a 80-95% inhibition of PGE2 production using TNFα-stimulated chondrocyte culture, indicating inhibition of inflammation. Microspheres loaded with the near infrared marker NIR780-iodide injected in healthy rat joints or joints with mild collagenase-induced OA showed retention of the microspheres up till 70days after injection. After intra-articular injection of TAA-loaded microspheres, TAA was detectable in the serum until day seven. Synovial inflammation was significantly lower in OA joints injected with TAA-loaded microspheres based on histological Krenn scores. Injection of TAA-loaded nor empty microspheres had no effect on cartilage integrity as determined by Mankin scoring. In conclusion, the PEA platform shows safety and efficacy upon intra-articular injection, and its extended degradation and release profiles compared to the currently used PLGA platforms may render it a good alternative. Even though further in vivo studies may need to address dosing and readout parameters such as pain, no effect on cartilage pathology was found and inflammation was effectively lowered in OA joints.

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.

Objective 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. Methods 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. Results 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. Conclusion 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.

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 x 106 and 10 x 106 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 x 106 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. Copyright (c) 2011 John Wiley & Sons, Ltd.

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.

Objective. 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. Methods. 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. Results. 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 x 10(-3) mm(3) in control joints versus 2.52 +/- 0.69 x 10(-3) mm(3) 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 mu m(3)/mu m(2) per day versus 0.30 +/- 0.03 mu m(3)/mu m(2) per day). Conclusion. 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.

Bone marrow-derived mesenchymal stem cells (MSCs) have demonstrated potential for regenerative medicine strategies. Knowledge of the way these cells respond to their environment in in vitro culture and after implantation in vivo is crucial for successful therapy. Oxygen tension plays a pivotal role in both situations. In vivo, a hypoxic environment can lead to apoptosis, but hypoxic preconditioning of MSCs and overexpression of prosurvival genes like Akt can reduce hypoxia-induced cell death. In cell culture, hypoxia can increase proliferation rates and enhance differentiation along the different mesenchymal lineages. Hypoxia also modulates the paracrine activity of MSCs, causing upregulation of various secretable factors, among which are important angiogenic factors such as vascular endothelial growth factor and interleukin-6 (IL6). Finally, hypoxia plays an important role in mobilization and homing of MSCs, primarily by its ability to induce stromal cell-derived factor-1 expression along with its receptor CXCR4. This article reviews the current literature on the effects of hypoxia on MSCs and aims to elucidate its potential role in regenerative medicine strategies.

Vascularization is still one of the most important limitations for the survival of engineered tissues after implantation. In this study, we aim to improve the in vivo vascularization of engineered adipose tissue by preforming vascular structures within in vitro-engineered adipose tissue constructs that can integrate with the host vascular system upon implantation. Different cell culture media were tested and different amounts of human adipose tissue-derived mesenchymal stromal cells (ASC) and human umbilical vein endothelial cells (HUVEC) were combined in spheroid cocultures to obtain optimal conditions for the generation of prevascularized adipose tissue constructs. Immunohistochemistry revealed that prevascular structures were formed in the constructs only when 20% ASC and 80% HUVEC were combined and cultured in a 1:1 mixture of endothelial cell medium and adipogenic medium. Moreover, the ASC in these constructs accumulated lipid and expressed the adipocyte-specific gene fatty acid binding protein-4. Implantation of prevascularized ASC/HUVEC constructs in nude mice resulted in a significantly higher amount of vessels (37 ± 17 vessels/mm2) within the constructs compared to non-prevascularized constructs composed only of ASC (3 ± 4 vessels/mm2). Moreover, a subset of the preformed human vascular structures (3.6 ± 4.2 structures/mm2) anastomosed with the mouse vasculature as indicated by the presence of intravascular red blood cells. Our results indicate that preformed vascular structures within in vitro-engineered adipose tissue constructs can integrate with the host vascular system and improve the vascularization upon implantation.

Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

Since the first cell therapeutic study to repair articular cartilage defects in the knee in 1994, several clinical studies have been reported. An overview of the results of clinical studies did not conclusively show improvement over conventional methods, mainly because few studies reach level I of evidence for effects on middle or long term. However, these explorative trials have provided valuable information about study design, mechanisms of repair and clinical outcome and have revealed that much is still unknown and further improvements are required. Furthermore, cellular and molecular studies using new technologies such as cell tracking, gene arrays and proteomics have provided more insight in the cell biology and mechanisms of joint surface regeneration. Besides articular cartilage, cartilage of other anatomical locations as well as progenitor cells are now considered as alternative cell sources. Growth Factor research has revealed some information on optimal conditions to support cartilage repair. Thus, there is hope for improvement. In order to obtain more robust and reproducible results, more detailed information is needed on many aspects including the fate of the cells, choice of cell type and culture parameters. As for the clinical aspects, it becomes clear that careful selection of patient groups is an important input parameter that should be optimized for each application. In addition, the study outcome parameters should be improved. Although reduced pain and improved function are, from the patient's perspective, the most important outcomes, there is a need for more structure/tissue-related outcome measures. Ideally, criteria and/or markers to identify patients at risk and responders to treatment are the ultimate goal for these more sophisticated regenerative approaches in joint surface repair in particular, and regenerative medicine in general.

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations. (C) 2008 Elsevier Inc. All rights reserved.

Transforming growth factor beta (TGF beta) is often used in cartilage tissue engineering to increase matrix formation by cells with various phenotypes. However, adverse effects of TGF beta, such as extensive cross-linking in cultured fibroblasts, have also been reported. Our goal was to study effects of TGF beta on collagen cross-linking and evaluating the role of cellular phenotype and physical environment. We therefore used four different cell populations in two very different physical environments: primary and expanded chondrocytes and fibroblasts embedded in alginate gel and attached to tissue culture plastic. Matrix production, collagen cross-linking, and alpha-smooth muscle actin (alpha SMA) were analyzed during 4 weeks with or without 2.5 ng/mL TGF beta 2. TGF beta 2 did not affect collagen deposition by primary cells. In expanded cells, TGF beta 2 increased collagen deposition. Chondrocytes and fibroblasts in monolayer produced more collagen cross-links with TGF beta 2. In alginate, primary and expanded cells displayed an unexpected decrease in collagen cross-linking with TGF beta 2. aSMA was not present in alginate cultures and barelay upregulated by TGF beta 2. Organized alpha SMA fibers were present in all monolayer cultures and became more pronounced with TGF beta 2. This study demonstrates that the physical environment determined by the substrate used co-determines the response of cells to TGF beta. The presence of mechanical stress, determined with alpha SMA-staining, is probably responsible for the increase in collagen cross-linking upon addition of TGF beta.

Human bone marrow-derived mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies, but loss of expansion and differentiation potential in vitro limits their applicability. Recently we showed that WNT3A protein promoted MSC proliferation and enhanced their chondrogenic potential, while simultaneously suppressing the propensity of the cartilage to undergo hypertrophic maturation. Since WNT3A protein is costly and rapidly loses its activity in culture, we investigated the possibility of replacing it with cheaper commercially available WNT agonists, specifically lithium chloride (LiCl), CHIR99021 (CHIR), SKL2001, and AMBMP. Of these, we found that only CHIR and LiCl stimulated MSC proliferation. Moreover, CHIR enhanced the chondrogenic capacity of MSCs, whereas LiCl predominantly increased the osteo- and adipogenic capacity. The different WNT agonists also differentially impacted the surface marker profile of the MSCs, possibly explaining the observed differences. Moreover, CHIR suppressed the hypertrophic propensity of the MSC-derived cartilage after in vivo implantation to an extent approaching that of WNT3A protein. These results indicate that CHIR may be a promising alternative for WNT3A protein for certain applications of human bone marrow-derived MSCs.

Objectives: Surgical manipulation of cartilage tissue is associated with chondrocyte death in the wound edges that hinders integration. The objective of this study was to evaluate the effect of a short course of treatment of a cartilage graft with a combination of hyaluronidase and collagenase on chondrocyte density and integrative capacity. Methods: Cartilage explants were treated with enzymes for various time periods and at various concentrations. A central core was punched out of a larger explant, treated with enzymes, reimplanted, and placed subcutaneously in athymic mice. The number of chondrocytes in the wound edges was counted, and the integrative capacity of the grafts was evaluated by histology. Results: Treatment with collagenase for 48 hours led to a significant increase in the number of vital chondrocytes and restored it to normal after 14 days of culture. Treatment with hyaluronidase and collagenase for 48 hours further increased chondrocyte densities to supranormal values. Shortening the treatment to 1 hour restored the chondrocyte density to normal after 14 days of culture. In vivo integration experiments showed increased chondrocyte densities in treated wound edges and extracellular matrix fibers crossing over from enzyme-treated parts to untreated parts. Conclusions: Short-duration treatment of a cartilage graft with a combination of hyaluronidase and collagenase increases cell density at wound edges and promotes integrative repair.

Summary Objective: An anterior cricoid split (ACS) causes an immediate distortion of the cricoid cartilage resulting in an anterior gap due to retraction of the cut ends. The objective of this animal study is to investigate: (1) to what extent the distortion after ACS is influenced by non-cartilaginous structures like tunica elastica, membranes, ligaments and muscles, which are connected to the cricoid; (2) how distortion is changing with further development; (3) in what way the distortion is affected by scoring of the internal surface of the cricoid; and (4) whether an immediate or late injury-induced distortion is related to age. Methods: Surgical interventions were performed in 20 young (8 weeks of age, 1300–1600 g) and 5 adult (28 weeks of age, 3500–4000 g) New Zealand White rabbits. The immediate effects were measured, and then the animals were followed for 20 weeks to study the long-term effects of the various procedures. Results: (1) The gap, immediately following an ACS, increased after additional transection of the cricothyroid ligament and the cricotracheal membrane, and even more when the cricovocal membrane was elevated from the inner surface of the cricoid arch. (2) The degree of distortion after various interventions in young animals appeared to increase substantially during further growth. (3) When the above-mentioned successive surgical steps were combined with scoring of the internal surface of the cricoid arch, a marked malformation of the split cricoid did develop with warping of the cut ends in lateral direction and a latero-cephalic rotation, the latter due to the action of the cricothyroid muscles. (4) The immediate distortion appeared to be similar in young and adult animals. During a follow-up of 20 weeks, a progressive distortion of the split cricoid ring was observed in the young growing rabbits. In adult animals, no significant progression of the distortion was found. Conclusions: The immediate and long-term distortion of the split cricoid is determined by the release of intrinsic forces of the cartilage, and extrinsic forces from non-cartilaginous structures like ligaments, muscles, membranes and tunica elastica.

Cartilage can be shaped by scoring. In an exploratory study in living adult animals, this phenomenon was demonstrated in cartilage of the nasal septum. Bending was observed immediately after superficial scoring of the cartilage surface, and the cartilage always warped in the direction away from the scored side. The scored piece of cartilage still showed its initially distorted shape 10 weeks after primary surgery. In ex vivo experiments, a clear relation between incision depth and bending of septal cartilage was observed. Under these controlled conditions, the variation between different septa was small. Deformation of the septal specimens was increased by introducing single superficial incisions deepening to half the thickness of the cartilage. A positive correlation between incision depth and bending was demonstrated. A model was used to accurately predict the degree of bending of the cartilage after making an incision of a particular depth. Hence, the effect of cartilage scoring can be predicted. Because the results of this controlled study showed excellent reproducibility for different septa, it is expected that this model can be extrapolated to human nasal septum cartilage. This would enable the surgeon to better predict the result of cartilage scoring, either preoperatively or perioperatively.

Summary Mesenchymal stem cells (MSCs) are a potential source of chondrogenic cells for the treatment of cartilage disorders, but loss of chondrogenic potential during in vitro expansion and the propensity of cartilage to undergo hypertrophic maturation impede their therapeutic application. Here we report that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion of human bone marrow-derived MSCs. The cells retained their chondrogenic potential and other phenotypic and functional properties of multipotent MSCs, which were gradually lost in the absence of WNT3A. Moreover, we discovered that endogenous WNT signals are the main drivers of the hypertrophic maturation that follows chondrogenic differentiation. Inhibition of WNT signals during differentiation prevented calcification and maintained cartilage properties following implantation in a mouse model. By maintaining potency during expansion and preventing hypertrophic maturation following differentiation, the modulation of WNT signaling removes two major obstacles that impede the clinical application of MSCs in cartilage repair., Highlights • WNT3A and FGF2 synergistically promote MSC proliferation • WNT3A and FGF2 synergistically enhance MSC chondrogenic potential during expansion • WNT3A and FGF2 maintain MSC characteristics over multiple passages • In vitro WNT signaling modulation leads to stable cartilage formation in vivo, van Osch, ten Berge, and colleagues show that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion and enhances the chondrogenic capacity of human bone marrow-derived MSCs. Moreover, inhibition of WNT signals during differentiation prevents hypertrophic maturation and calcification in vivo, until now one of the major obstacles in the use of MSCs for cartilage repair.

Background: Cartilage tissue engineering can offer promising solutions for restoring cartilage defects in the head and neck area and has the potential to overcome limitations of current treatments. However, to generate a construct of reasonable size, large numbers of chondrocytes are required, which limits its current applicability. Therefore, the authors evaluate the suitability of a combination of cells for cartilage regeneration: bone marrow-derived mesenchymal stem cells and ear or nasal chondrocytes. Methods: Human bone marrow-derived mesenchymal stem cells were encapsulated in alginate hydrogel as single-cell-type populations or in combination with bovine ear chondrocytes or nasal chondrocytes at an 80:20 ratio. Constructs were either cultured in vitro or implanted directly subcutaneously into mice. Cartilage formation was evaluated with biochemical and biomechanical analyses. The use of a xenogeneic coculture system enabled the analyses of the contribution of the individual cell types using species-specific gene-expression analyses. Results: In vivo, human bone marrow-derived mesenchymal stem cells/bovine ear chondrocytes or human bone marrow-derived mesenchymal stem cells/bovine nasal chondrocytes contained amounts of cartilage components similar to those of constructs containing chondrocytes only (i.e., bovine ear and nasal chondrocytes). In vitro, species-specific gene-expression analyses demonstrated that aggrecan was expressed by the chondrocytes only, which suggests a more trophic role for human bone marrow-derived mesenchymal stem cells. Furthermore, the additional effect of human bone marrow-derived mesenchymal stem cells was more pronounced in combination with bovine nasal chondrocytes. Conclusions: By supplementing low numbers of bovine ear or nasal chondrocytes with human bone marrow-derived mesenchymal stem cells, the authors were able to engineer cartilage constructs with properties similar to those of constructs containing chondrocytes only. This makes the procedure more feasible for future applicability in the reconstruction of cartilage defects in the head and neck area because fewer chondrocytes are required. CLINICAL QUESTIONS/LEVEL OF EVIDENCE: Therapeutic, V.

Cartilage structures from the head and neck possess a certain but limited capacity to heal after injury. This capacity is accredited to the perichondrium. In this study, the role of the inner (cambium) and the outer (fibrous) layers of the perichondrium in cartilage wound healing in vitro is investigated. For the first time, the possibility of selectively removing the outer perichondrium layer is presented. Using rabbit ears, three different conditions were created: cartilage explants with both perichondrium layers intact, cartilage explants with only the outer perichondrium layer dissected, and cartilage explants with both perichondrium layers removed. The explants were studied after 0, 3, 7, 14, and 21 days of in vitro culturing using histochemistry and immunohistochemistry for Ki-67, collagen type II, transforming growth factor beta 1 (TGFbeta1), and fibroblast growth factor 2 (FGF2). When both perichondrium layers were not disturbed, fibrous cells grew over the cut edges of the explants from day 3 of culture on. New cartilage formation was never observed in this condition. When only the outer perichondrium layer was dissected from the cartilage explants, new cartilage formation was observed around the whole explant at day 21. When both perichondrium layers were removed, no alterations were observed at the wound surfaces. The growth factors TGFbeta1 and FGF2 were expressed in the entire perichondrium immediately after explantation. The expression gradually decreased with time in culture. However, the expression of TGFbeta1 remained high in the outer perichondrium layer and the layer of cells growing over the explant. This indicates a role for TGFbeta1 in the enhancement of fibrous overgrowth during the cartilage wound-healing process. The results of this experimental in vitro study demonstrate the dual role of perichondrium in cartilage wound healing. On the one hand, the inner layer of the perichondrium, adjacent to the cartilage, provides (in time) cells for new cartilage formation. On the other hand, the outer layer rapidly produces fibrous overgrowth, preventing the good cartilage-to-cartilage connection necessary to restore the mechanical function of the structure.

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.

The use of a composite graft of bovine trabecular demineralized bone matrix (DBM) and perichondrium has been found a reliable method for in vivo generation of cartilage. In the present study, the mechanism whereby this commercially available matrix increases cartilage formation was investigated. First, the time course of cartilage formation in vivo, in the combined implant of perichondrium and DBM in the rabbit ear was studied, with special focus on tissue reactions to DBM. DBM was colonized by macrophages from day 3 post-operatively, reaching a maximum after 2 weeks. Only a minimal number of neutrophils was found. After 3 weeks the DBM appeared to be resorbed. In the first week the DBM was invaded with chondroblasts, and chondrogenesis occurred between the first and second week of implantation. After 3 weeks, the initially formed islets of cartilage had fused. Next, the chondrogenic capacity of DBM itself was investigated by implantation of DBM without perichondrium. This never resulted in cartilage formation. Immunohistochemistry showed only a faint staining of the DBM for growth factors. This indicates a minimal chondrogenic e⁄ect of DBM alone and the requirement of perichondrium as cell provider. In order to define the conditions which cause chondrogenesis in composites of perichondrium and DBM, a series of in vitro culture experiments was performed in which the in vivo situation was mimicked step by step. The basic condition was perichondrium cultured in medium with 10% FCS. In this condition, cartilage formation was variable. Because in the in vivo situation both DBM and macrophages can release growth factors, the e⁄ect of IGF1, TGFb2 or OP1 added to the culture medium was tested. Neither the incidence nor the amount of cartilage formation was stimulated by addition of growth factors. Perichondrium wrapped around DBM in vitro gave cartilage formation in the perichondrium but the incidence and amount were not significantly stimulated compared to cultures of perichondrium without DBM. However, cartilage-like cells were found in the DBM suggesting an e⁄ect of DBM on perichondrium-derived cells. Finally, macrophages and/or blood were added to the composite DBM-perichondrium to mimic the in vivo situation as close as possible. However, no e⁄ect of this treatment was found. In conclusion, this study indicates that DBM itself has few chondrogenic qualities but functions merely as a spacer for cell ingrowth. The fast resorption of DBM by macrophages in vivo seems of importance for the cartilage forming process, but in vitro the presence of macrophages (in combination with blood) could not enhance chondrogenesis. ( 1999 Elsevier Science Ltd. All rights reserved

Tissue Engineering. Part B, Reviews, 21 (2), ISSN:1937-3368, ISSN:1937-3376

A pedicled auricular perichondrial flap wrapped around trabecular demineralized bovine bone matrix can generate an autologous cartilage graft. In earlier experimental studies, it was demonstrated that this graft could be used for nasal and cricoid reconstruction. It was assumed that the vascularization of the perichondrial flap was obligatory, but it was never proven that the flap should be pedicled. Moreover, for clinical use, the dimensions of the auricle would set restrictions to the size of the graft generated. Therefore, the possibility to generate cartilage with a composite graft of a free perichondrial flap wrapped around demineralized bovine bone matrix, by using young New Zealand White rabbits, was studied. This composite graft was implanted at poorly (subcutaneously in the abdominal wall; n = 12), fairly (subcutaneously in the pinna; n = 12), and well-vascularized sites (quadriceps muscle; n = 12). As a control, trabecular demineralized bovine bone matrix was implanted without perichondrial cover. Half of these grafts (n = 6) were harvested after 3 weeks, and the remaining grafts (n = 6) after 6 weeks of implantation. In histologic sections of these grafts, the incidence of cartilage formation was scored. Furthermore, the amount of newly formed cartilage was calculated by computerized histomorphometry. Trabecular demineralized bovine bone matrix without perichondrial cover demonstrated early resorption; no cartilage or bone was formed. In demineralized bovine bone matrix wrapped in perichondrium, early cartilage formed after 3 weeks at well- and fairly vascularized sites. No cartilage could be detected in grafts placed at a poorly vascularized site after 3 weeks; minimal cartilage formed after 6 weeks. In summary, the highest incidence of cartilage formed when trabecular demineralized bovine bone matrix was wrapped either in a pedicled auricular perichondrial flap or in a free perichondrial flap, which was placed at a well-vascularized site. Second, a significantly higher percentage of the total area of the graft was cartilaginized at well-vascularized sites after 3 weeks. The newly generated cartilage contained collagen type II and proteoglycans with hyaluronic acid binding regions, whereas collagen type I was absent, indicating the presence of hyaline cartilage. This study demonstrates that new cartilage suitable for a graft can be generated by free perichondrial flaps, provided that the site of implantation is well vascularized. Consequently, the size of such a graft is no longer limited to the dimensions of the auricle.

This study was performed to determine the various processes involved in the behaviour of hyaline cartilage during the wound healing period after trauma or surgery of vulnerable structures like the nasal septal cartilage and the cricoid. The results of different procedures (perpendicular and parallel to the cartilage surface) in young and young-adult animals were analyzed: septal incision at different locations (young-old), cricoid split (young-old), suturing cartilage, closing defects with autologous cartilage (young), biomaterials (young) and newly engineered cartilage in 4- and 24-week-old rabbits (series of ten animals). Cartilage of the young rabbit and child have similar hyaline cartilage with a varying distribution in thickness. Thinner areas are more susceptible to malformations. Incisions through younger cartilage give rise to some new cartilage formation covered by a new layer of perichondrium: through older, differentiated cartilage the incision causes superficial but permanent necrosis. Edges of cut cartilage mostly do heal by formation of fibrous junctions. This forms a weak spot, sensitive to deviations. The same fate goes for the healing between the autologous graft and the surrounding pre-existent cartilage. Trauma parallel to the surface, leads to inconsistent quantity of neocartilage. With ageing the wound healing and regenerative capacities decrease. In general, biomaterials are less accepted by the surrounding tissues and would impede further growth. Only newly engineered, and thus less differentiated (younger) cartilage of hyaline nature, appeared to be well accepted at the interface with the edges of a cartilage defect. There are indications that the release of growth factors might play a role in cartilage wound healing. In the child as well as the adult, wound healing of hyaline cartilage structures is incomplete, and surgery remains 'experimental' surgery. The clinical implications of gradual loss of the regenerative capacity of hyaline cartilage should be further investigated.

Knowledge of matrix assembly is necessary to understand the pathogenesis of disease processes and to find solutions for repair of articular cartilage lesions. The influence of growth factors on matrix assembly is largely unknown. We investigated whether, and to what degree, insulin-like growth factor (IGF-1) and transforming growth factor beta-2 (TGF beta-2) influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment (consisting of pericellular and territorial matrix) compared to the further-removed matrix compartment (consisting of the interterritorial matrix).Bovine articular chondrocytes were cultured in alginate beads for day 13. The effect of addition of 25 ng/ml IGF-1 or 25 ng/ml TGF beta-2 during the last 7 days in culture was determined. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment. The amount of DNA, the total amount of proteoglycans and the amount of newly synthesized proteoglycans were analyzed biochemically. Morphometric analysis on electron micrographs was used to calculate the volumes of the cell-associated and further-removed matrix components.It was demonstrated in control beads that 25 +/- 8% of the proteoglycans were laid down in the cell-associated matrix compartment compared with 75 +/- 8% in the further-removed matrix compartment. The cell-associated matrix compartment in intact beads could be recognized in electron microscopy by a delineation of dense amorph material. Morphometric evaluation showed a relative volume of the cell-associated matrix compartment of 5.2 +/- 0.6% compared with 91.3 +/- 0.8% of the further-removed matrix compartment and 3.5 +/- 0.3% of the area occupied by cells. Combination of biochemical and morphometric results showed that the concentration of proteoglycans in the cell-associated matrix compartment was 3.63 +/- 0.32 mg/ml. By adding IGF-1 or TGF beta-2, the amount of both total accumulated proteoglycans and newly synthesized [35S]proteoglycans at day 13 in culture increased. In addition to an overall rise in proteoglycan content, IGF-1 significantly increased (24%) the percentage of proteoglycans laid down in the cell-associated matrix compartment while not changing the relative volume of this compartment (5.2 +/- 0.8%). This leads to a 82% (P0.05) increase in the proteoglycan concentration in the cell-associated matrix compartment compared to control beads. In contrast, TGF beta-2 significantly decreased (24%) the relative amount of proteoglycans in the cell-associated matrix compartment which was paralleled by a reduction of the relative volume from 5.2 +/- 0.6 to 3.6 +/- 1.4%. This leads to a significant increase of 87% of the proteoglycan concentration in the cell-associated matrix compartment.This study demonstrates that both IGF-1 and TGF beta-2 significantly but differently influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment of cultured bovine chondrocytes in alginate. Both growth factors increase the concentration of proteoglycans in the cell-associated matrix compartment. However, addition of TGF beta-2 to bovine articular chondrocytes cultured in alginate beads for 13 days results in a significant reduction of the relative volume of the pericellular matrix compartment compared to controls, indicating differences in assembly of the matrix.

Item does not contain fulltext In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitro chondrogenesis, it was found that supplementing transforming growth factor beta (TGFbeta) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGFbeta; however, the observed results could not be attributed to TGFbeta, as either capturing TGFbeta with an antibody or blocking the canonical TGFbeta signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGFbeta family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role.

Introduction Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration. Methods sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness. Results All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation. Conclusions Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced subchondral sclerosis, synovial macrophage activation, and osteophyte formation., Arthritis Research & Therapy, 16 (1), ISSN:1465-9905, ISSN:1465-9913, ISSN:1478-6362, ISSN:1478-6354

Success of adipose tissue engineering for soft tissue repair has been limited by insufficient adipogenic differentiation, an unfavorable host response, and insufficient vascularization. In this study, we examined how scaffold-free spheroid and fibrin-based environments impact these parameters in human adipose-derived stromal cell (ASC)-based adipose constructs. ASCs were differentiated in spheroids or fibrin-based constructs. After 7 days, conditioned medium was collected and spheroids/fibrin-based constructs were either harvested or implanted subcutaneously in athymic mice. Following 7 days of implantation, the number of blood vessels in fibrin-based constructs was significantly higher than in spheroids (93 ± 45 vs. 23 ± 11 vessels/mm2) and the inflammatory response to fibrin-based constructs was less severe. The reasons for these results were investigated further in vitro. We found that ASCs in fibrin-based constructs secreted significantly higher levels of the angiogenic factors VEGF and HGF and lower levels of the inflammatory cytokine IL-8. Furthermore, ASCs in fibrin-based constructs secreted significantly higher levels of leptin and showed a 2.5-fold upregulation of the adipogenic transcription factor PPARG and a fourfold to fivefold upregulation of the adipocyte-specific markers FABP4, perilipin, and leptin. These results indicate that fibrin-based ASC constructs are potentially more suitable for ASC-based adipose tissue reconstruction than scaffold-free spheroids.

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.

Background 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. Methods 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. Results 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. Conclusions 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.

Laryngotracheal reconstruction requires a supportive structure with a mucosal lining, which needs a vascular supply in order to regenerate properly. We investigated the necessity of a vascular carrier and mucosal graft when using porous titanium for laryngotracheal reconstruction. Surgical defects of the laryngotracheal complex in 22 rabbits were reconstructed with: (a) porous titanium implanted on a vascularized fascia combined with a buccal mucosal graft (first stage) before transposing to the neck area (second stage); (b) porous titanium implanted on a vascularized fascia (first stage) combined with a mucosal graft (second stage); (c) porous titanium on a pedicled fascia flap; and (d) porous titanium alone. The grafts were tolerated well. Re-epithelialization occurred in all groups. Normal mucosa with a submucosal layer containing vital cells was noted using the titanium implants. Blood vessels were grown in the pores of the titanium scaffold to supply the overlying mucosa. The scaffold was well integrated in the adjacent tracheal cartilage and surrounding tissues, except in the two cases that showed titanium displacement. Inflammation and granulation formation were seen in most rabbits in groups III and IV, initiated probably by the use of buccal mucosal grafts. Reconstruction of a rabbit's trachea using composites of porous titanium, mucosal grafts and a fascia flap is feasible. Titanium seems to meet the requirements needed for closing a small defect of the tracheal wall and allows for re-epithelialization. For larger defects, a vascular carrier with a mucosal graft is probably indispensable to ensure the process of re-epithelialization. Copyright (C) 2010 John Wiley & Sons, Ltd.