Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair.

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100-150 µm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair.

Bone formation by human paediatric marrow stromal cells in a functional allogeneic immune system.

Allogeneic stem-cell based regenerative medicine is a promising approach for bone defect repair. The use of chondrogenically differentiated human marrow stromal cells (MSCs) has been shown to lead to bone formation by endochondral ossification in immunodeficient pre-clinical models. However, an insight into the interactions between the allogeneic immune system and the human MSC-derived bone grafts has not been fully achieved yet. The choice of a potent source of MSCs isolated from pediatric donors with consistent differentiation and high proliferation abilities, as well as low immunogenicity, could increase the chance of success for bone allografts. In this study, we employed an immunodeficient animal model humanised with allogeneic immune cells to study the immune responses towards chondrogenically differentiated human pediatric MSCs (ch-pMSCs). We show that ch-differentiated pMSCs remained non-immunogenic to allogeneic CD4 and CD8 T cells in an in vitro co-culture model. After subcutaneous implantation in mice, ch-pMSC-derived grafts were able to initiate bone mineralisation in the presence of an allogeneic immune system for 3 weeks without the onset of immune responses. Re-exposing the splenocytes of the humanised animals to pMSCs did not trigger further T cell proliferation, suggesting an absence of secondary immune responses. Moreover, ch-pMSCs generated mature bone after 8 weeks of implantation that persisted for up to 6 more weeks in the presence of an allogeneic immune system. These data collectively show that human allogeneic chondrogenically differentiated pediatric MSCs might be a safe and potent option for bone defect repair in the tissue engineering and regenerative medicine setting.

Addition of heparin binding sites strongly increases the bone forming capabilities of BMP9 in vivo.

Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9's mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential.

Effectiveness of BMP-2 and PDGF-BB Adsorption onto a Collagen/Collagen-Magnesium-Hydroxyapatite Scaffold in Weight-Bearing and Non-Weight-Bearing Osteochondral Defect Bone Repair: In Vitro, Ex Vivo and In Vivo Evaluation.

Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.

Swelling-Dependent Shape-Based Transformation of a Human Mesenchymal Stromal Cells-Laden 4D Bioprinted Construct for Cartilage Tissue Engineering.

3D bioprinting is usually implemented on flat surfaces, posing serious limitations in the fabrication of multilayered curved constructs. 4D bioprinting, combining 3D bioprinting with time-dependent stimuli-induced transformation, enables the fabrication of shape-changing constructs. Here, a 4D biofabrication method is reported for cartilage engineering based on the differential swelling of a smart multi-material system made from two hydrogel-based materials: hyaluronan and alginate. Two ink formulations are used: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Both inks have similar elastic, shear-thinning, and printability behavior. The inks are 3D printed into a bilayered scaffold before triggering the shape-change by using liquid immersion as stimulus. In time (4D), the differential swelling between the two zones leads to the scaffold's self-bending. Different designs are made to tune the radius of curvature and shape. A bioprinted formulation of AHAT and human bone marrow cells demonstrates high cell viability. After 28 days in chondrogenic medium, the curvature is clearly present while cartilage-like matrix production is visible on histology. A proof-of-concept of the recently emerged technology of 4D bioprinting with a specific application for the design of curved structures potentially mimicking the curvature and multilayer cellular nature of native cartilage is demonstrated.

Intra-articular Administration of Triamcinolone Acetonide in a Murine Cartilage Defect Model Reduces Inflammation but Inhibits Endogenous Cartilage Repair.

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.

Intra-articular injection of triamcinolone acetonide sustains macrophage levels and aggravates osteophytosis during degenerative joint disease in mice.

BACKGROUND AND PURPOSE: Corticosteroids such as triamcinolone acetonide (TAA) are potent drugs administered intra-articularly as an anti-inflammatory therapy to relieve pain associated with osteoarthritis (OA). However, the ability of early TAA intervention to mitigate OA progression and modulate immune cell subsets remains unclear. Here, we sought to understand the effect of early intra-articular injection of TAA on OA progression, local macrophages, and peripheral blood monocytes. EXPERIMENTAL APPROACH: Degenerative joint disease was induced by intra-articular injection of collagenase into the knee joint of male C57BL/6 mice. After 1 week, TAA or saline was injected intra-articularly. Blood was taken throughout the study to analyse monocyte subsets. Mice were killed at days 14 and 56 post-induction of collagenase-induced OA (CiOA) to examine synovial macrophages and structural OA features. KEY RESULTS: The percentage of macrophages relative to total live cells present within knee joints was increased in collagenase- compared with saline-injected knees at day 14 and was not altered by TAA treatment. However, at day 56, post-induction of CiOA, TAA-treated knees had increased levels of macrophages compared with the knees of untreated CiOA-mice. The distribution of monocyte subsets present in peripheral blood was not altered by TAA treatment during the development of CiOA. Osteophyte maturation was increased in TAA-injected knees at day 56. CONCLUSION AND IMPLICATIONS: Intra-articular injection of TAA increases long-term synovial macrophage numbers and osteophytosis. Our findings suggest that TAA accentuates the progression of osteoarthritis-associated features when applied to an acutely inflamed knee.

Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides.

Antisense oligonucleotides (ASOs) carry an enormous therapeutic potential in different research areas, however, the lack of appropriate carriers for their delivery to the target tissues is hampering their clinical translation. The present study investigates the application of novel biomimetic nano-vesicles, Nano-Ghosts (NGs), for the delivery of ASOs to human mesenchymal stem cells (MSCs), using a microRNA inhibitor (antimiR) against miR-221 as proof-of-concept. The integration of this approach with a hyaluronic acid-fibrin (HA-FB) hydrogel scaffold is also studied, thus expanding the potential of NGs applications in regenerative medicine. The study shows robust antimiR encapsulation in the NGs using electroporation and the NGs ability to be internalized in MSCs and to deliver their cargo while avoiding endo-lysosomal degradation. This leads to rapid and strong knock-down of miR-221 in hMSCs in vitro, both in 2D and 3D hydrogel culture conditions (>90% and > 80% silencing efficiency, respectively). Finally, in vivo studies performed with an osteochondral defect model demonstrate the NGs ability to effectively deliver antimiR to endogenous cells. Altogether, these results prove that the NGs can operate as stand-alone system or as integrated platform in combination with scaffolds for the delivery of ASOs for a wide range of applications in drug delivery and regenerative medicine.

Macrophage phenotypes and monocyte subsets after destabilization of the medial meniscus in mice.

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 mice compared with 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 coexistence with joint instability may be a requirement to initiate and exacerbate OA progression.

MSC encapsulation in alginate microcapsules prolongs survival after intra-articular injection, a longitudinal in vivo cell and bead integrity tracking study.

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. Graphical Abstract.

CD4+ CCR6+ T cells, but not γδ T cells, are important for the IL-23R-dependent progression of antigen-induced inflammatory arthritis in mice.

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R+ T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23RGFP/+ ) mice, that CD4+ CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP)+ CD4+ CCR6+ T cells, but not IL-23R(GFP)+ γδ T cells, were present in the inflamed joints. IL-23RGFP/+ mice were bred as homozygotes to obtain IL-23RGFP/GFP (IL-23R deficient/IL-23R-/- ) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R-/- mice, which revealed less IL-17A+ cells in their lymphoid tissues. Surprisingly, IL-23R-/- mice had increased numbers of IL-23R(GFP)+ CD4+ CCR6+ and CCR7+ CD4+ CCR6+ T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP)+ CD4+ CCR6+ T cells were present in the synovium of IL-23R-/- mice at day 4. Finally, adoptive transfer experiments revealed that CD4+ CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4+ CCR6+ T cells and not on γδ T cells.

Hydrogel-based delivery of antimiR-221 enhances cartilage regeneration by endogenous cells.

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.

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

Human Mesenchymal Stromal Cell Sheets Induce Macrophages Predominantly to an Anti-Inflammatory Phenotype.

Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that mesenchymal stromal cells can modulate macrophage-induced inflammation and, therefore, are promising candidates for cell-based therapies aimed at promoting tissue repair. Recently, cell sheets were introduced as a new method of delivering stromal cells to the repair site. The goal of the current study was to compare the effect of different types of stromal cell sheets on the inflammatory state of macrophages in vitro. We compared the effects of adipose tissue-derived stromal cell (ASC) sheets, bone marrow derived stromal cell (BMSC) sheets, and fibroblast sheets on macrophage functional phenotype using flow cytometric analysis, gene expression, as well as cell sheet protein secretion. This was evaluated with and without inflammatory stimulation. Viability and senescence for the different types of sheet were also evaluated. Macrophages cultured in ASC sheet conditioned medium (CM) displayed a higher fluorescence intensity of the anti-inflammatory CD206 surface marker than when cultured in BMSC sheet CM and expressed more CCL18 and IL1RA than when cultured in fibroblast sheet CM. Moreover, ASC sheets had higher cell viability and less senescent cells than BMSC sheets and fibroblast sheets. Taken together, ASC and BMSC can stimulate the anti-inflammatory macrophage (M2) phenotype to a better extent than fibroblasts. It is suggested that ASC sheets might outperform BMSC sheets in an inflammatory situation since ASC sheet CM induced-macrophages have more M2 characteristics, and ASC in the sheet was more viable.

Intra-articular Injections of Platelet-Rich Plasma Releasate Reduce Pain and Synovial Inflammation in a Mouse Model of Osteoarthritis.

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.

Degradation, Intra-Articular Biocompatibility, Drug Release, and Bioactivity of Tacrolimus-Loaded Poly(d-l-lactide-PEG)-b-poly(l-lactide) Multiblock Copolymer-Based Monospheres.

The aim of this study was to develop a formulation with a sustained intra-articular release of the anti-inflammatory drug tacrolimus. Drug release kinetics from the prepared tacrolimus loaded monodisperse biodegradable microspheres based on poly(d-l-lactide-PEG)-b-poly(l-lactide) multiblock copolymers were tunable by changing polymer composition, particularly hydrophobic-hydrophilic block ratio. The monospheres were 30 µm and released the drug, depending on the formulation, in 7 to >42 days. The formulation exhibiting sustained release for 1 month was selected for further in vivo evaluation. Rat knees were injected with three different doses of tacrolimus (10 wt %) loaded monospheres (2.5, 5.0, and 10 mg), contralateral control knees with saline. Micro-CT and histology showed no negative changes on cartilage, indicating good biocompatibility. Minor osteophyte formation was seen in a dose dependent fashion, suggesting local drug release and therapeutic action thereof. To investigate in vivo drug release, tacrolimus monospheres were injected into horse joints, after which multiple blood and synovial fluid samples were taken. Sustained intra-articular release was seen during the entire four-week follow-up, with negligible systemic drug concentrations (<1 ng/mL), confirming the feasibility of local intra-articular drug delivery without provoking systemic effects. Intra-articular injection of unloaded monospheres led to a transient inflammatory reaction, measured by total synovial leucocyte count (72 h). This reaction was significantly lower in joints injected with tacrolimus loaded monospheres, showing not only the successful local tacrolimus delivery but also local anti-inflammatory action. This local anti-inflammatory potential without systemic side-effects can be beneficial in the treatment of inflammatory joint diseases, among which is osteoarthritis.

Effects of adipose stem cell sheets on colon anastomotic leakage in an experimental model: Proof of principle.

The most dreaded complication of colorectal surgery is anastomotic leakage. Adipose tissue-derived stem cell sheets (ASC sheets) prepared from temperature-responsive culture surfaces can be easily transplanted onto tissues. These sheets are proposed to improve cell transplant efficiency and enhance wound healing. The aim of this study was to investigate whether application of ASC sheets could prevent leakage of sutured colorectal anastomoses. Insufficient suturing of colorectal anastomoses was performed in Wistar rats to create a colorectal anastomotic leakage model. Rats were randomized to ASC sheet application or control group. Leakage, abscess formation, adhesion formation, anastomotic bursting pressure (ABP), and histology were evaluated on postoperative day 3 or 7. ASC sheet application significantly reduced anastomotic leakage compared to controls, without increased adhesion formation. ASC sheet transplantation resulted in more CD3+ T-cells and CD163+ anti-inflammatory macrophages at the anastomotic site than the control group. ABP, vessel density and collagen deposition were not different between groups. Using cell sheet technology, we generated ASC sheets that prevented disruption of sutured colorectal anastomoses as shown by reduced leakage. Increased numbers of anti-inflammatory macrophages and T-cells might have contributed to this positive effect.

Degradation, intra-articular retention and biocompatibility of monospheres composed of [PDLLA-PEG-PDLLA]-b-PLLA multi-block copolymers.

In this study, we investigated the use of microspheres with a narrow particle size distribution ('monospheres') composed of biodegradable poly(DL-lactide)-PEG-poly(DL-lactide)-b-poly(L-lactide) multiblock copolymers that are potentially suitable for local sustained drug release in articular joints. Monospheres with sizes of 5, 15 and 30µm and a narrow particle size distribution were prepared by a micro-sieve membrane emulsification process. During in vitro degradation, less crystallinity, higher swelling and accelerated mass loss during was observed with increasing the PEG content of the polymer. The monospheres were tested in both a small (mice/rat) and large animal model (horse). In vivo imaging after injection with fluorescent dye loaded microspheres in mice knees showed that monospheres of all sizes retained within the joint for at least 90days, while the same dose of free dye redistributed to the whole body within the first day after intra-articular injection. Administration of monospheres in equine carpal joints caused a mild transient inflammatory response without any clinical signs and without degradation of the cartilage, as evidenced by the absence of degradation products of sulfated glycosaminoglycans or collagen type 2 in the synovial fluid. The excellent intra-articular biocompatibility was confirmed in rat knees, where µCT-imaging and histology showed neither changes in cartilage quality nor quantity. Given the good intra-articular retention and the excellent biocompatibility, these novel poly(DL-lactide)-PEG-poly(DL-lactide)-b-poly(L-lactide)-based monospheres can be considered a suitable platform for intra-articular drug delivery. STATEMENT OF SIGNIFICANCE: This paper demonstrates the great potential in intra-articular drug delivery of monodisperse biodegradable microspheres which were prepared using a new class of biodegradable multi-block copolymers and a unique membrane emulsification process allowing the preparation of microspheres with a narrow particle size distribution (monospheres) leading to multiple advantages like better injectability, enhanced reproducibility and predictability of the in vivo release kinetics. We report not only on the synthesis and preparation, but also in vitro characterization, followed by in vivo testing of intra-articular biocompatibility of the monospheres in both a small and a large animal model. The favourable intra-articular biocompatibility combined with the prolonged intra-articular retention (>90days) makes these monospheres an interesting drug delivery platform. What should also be highlighted is the use of horses; a very accurate translational model for the human situation, making the results not only relevant for equine healthcare, but also for the development of novel human OA therapies.

High fat diet accelerates cartilage repair in DBA/1 mice.

Obesity is a well-known risk factor for osteoarthritis, but it is unknown what it does on cartilage repair. Here we investigated whether a high fat diet (HFD) influences cartilage repair in a mouse model of cartilage repair. We fed DBA/1 mice control or HFD (60% energy from fat). After 2 weeks, a full thickness cartilage defect was made in the trochlear groove. Mice were sacrificed, 1, 8, and 24 weeks after operation. Cartilage repair was evaluated on histology. Serum glucose, insulin and amyloid A were measured 24 h before operation and at endpoints. Immunohistochemical staining was performed on synovium and adipose tissue to evaluate macrophage infiltration and phenotype. One week after operation, mice on HFD had defect filling with fibroblast-like cells and more cartilage repair as indicated by a lower Pineda score. After 8 weeks, mice on a HFD still had a lower Pineda score. After 24 weeks, no mice had complete cartilage repair and we did not detect a significant difference in cartilage repair between diets. Bodyweight was increased by HFD, whereas serum glucose, amyloid A and insulin were not influenced. Macrophage infiltration and phenotype in adipose tissue and synovium were not influenced by HFD. In contrast to common wisdom, HFD accelerated intrinsic cartilage repair in DBA/1 mice on the short term. Resistance to HFD induced inflammatory and metabolic changes could be associated with accelerated cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1258-1264, 2017.

Silencing of Antichondrogenic MicroRNA-221 in Human Mesenchymal Stem Cells Promotes Cartilage Repair In Vivo.

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 toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic 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-ß. 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 toward chondrogenic differentiation in the absence of TGF-ß. 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 prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801-1811.