Necrosis reduction efficacy of subdermal biomaterial mediated oxygen delivery in ischemic skin flaps.

Inadequate tissue blood supply as may be found in a wound or a poorly vascularised graft, can result in tissue ischemia and necrosis. As revascularization is a slow process relative to the proliferation of bacteria and the onset of tissue necrosis, extensive tissue damage and loss can occur before healing is underway. Necrosis can develop rapidly, and treatment options are limited such that loss of tissue following necrosis onset is considered unavoidable and irreversible. Oxygen delivery from biomaterials exploiting aqueous decomposition of peroxy-compounds has shown some potential in overcoming the supply limitations by creating oxygen concentration gradients higher than can be attained physiologically or by air saturated solutions. We sought to test whether subdermal oxygen delivery from a material composite that was buffered and contained a catalyst, to reduce hydrogen peroxide release, could ameliorate necrosis in a 9 × 2 cm flap in a rat model that reliably underwent 40 % necrosis if untreated. Blood flow in this flap reduced from near normal to essentially zero, along its 9 cm length and subdermal perforator vessel anastomosis was physically prevented by placement of a polymer sheet. In the middle, low blood flow region of the flap, treatment significantly reduced necrosis based on measurements from photographs and histological micrographs. No change was observed in blood vessel density but significant differences in HIF1-α, inducible nitric oxide synthase and liver arginase were observed with oxygen delivery.

Development of Neovasculature in Axially Vascularized Calcium Phosphate Cement Scaffolds.

Augmenting the vascular supply to generate new tissues, a crucial aspect in regenerative medicine, has been challenging. Recently, our group showed that calcium phosphate can induce the formation of a functional neo-angiosome without the need for microsurgical arterial anastomosis. This was a preclinical proof of concept for biomaterial-induced luminal sprouting of large-diameter vessels. In this study, we investigated if sprouting was a general response to surgical injury or placement of an inorganic construct around the vessel. Cylindrical biocement scaffolds of differing chemistries were placed around the femoral vein. A contrast agent was used to visualize vessel ingrowth into the scaffolds. Cell populations in the scaffold were mapped using immunohistochemistry. Calcium phosphate scaffolds induced 2.7-3 times greater volume of blood vessels than calcium sulphate or magnesium phosphate scaffolds. Macrophage and vSMC populations were identified that changed spatially and temporally within the scaffold during implantation. NLRP3 inflammasome activation peaked at weeks 2 and 4 and then declined; however, IL-1ß expression was sustained over the course of the experiment. IL-8, a promoter of angiogenesis, was also detected, and together, these responses suggest a role of sterile inflammation. Unexpectedly, the effect was distinct from an injury response as a result of surgical placement and also was not simply a foreign body reaction as a result of placing a rigid bioceramic next to a vein, since, while the materials tested had similar microstructures, only the calcium phosphates tested elicited an angiogenic response. This finding then reveals a potential path towards a new strategy for creating better pro-regenerative biomaterials.

In Vitro Cellular and Molecular Interplay between Human Foreskin-Derived Mesenchymal Stromal/Stem Cells and the Th17 Cell Pathway.

Foreskin, considered a biological waste material, has been shown to be a reservoir of therapeutic cells. The immunomodulatory properties of mesenchymal stromal/stem cells (MSCs) from the foreskin (FSK-MSCs) are being evaluated in cell-based therapy for degenerative, inflammatory and autoimmune disorders. Within the injured/inflamed tissue, proinflammatory lymphocytes such as IL-17-producing T helper cells (Th17) may interact with the stromal microenvironment, including MSCs. In this context, MSCs may encounter different levels of T cells as well as specific inflammatory signals. Uncovering the cellular and molecular changes during this interplay is central for developing an efficient and safe immunotherapeutic tool. To this end, an in vitro human model of cocultures of FSK-MSCs and T cells was established. These cocultures were performed at different cell ratios in the presence of an inflammatory setting. After confirming that FSK-MSCs respond to ISCT criteria by showing a typical phenotype and multilineage potential, we evaluated by flow cytometry the expression of Th17 cell markers IL-17A, IL23 receptor and RORγt within the lymphocyte population. We also measured 15 human Th17 pathway-related cytokines. Regardless of the T cell/MSC ratio, we observed a significant increase in IL-17A expression associated with an increase in IL-23 receptor expression. Furthermore, we observed substantial modulation of IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α secretion. These findings suggest that FSK-MSCs are receptive to their environment and modulate the T cell response accordingly. The changes within the secretome of the stromal and immune environment are likely relevant for the therapeutic effect of MSCs. FSK-MSCs represent a valuable cellular product for immunotherapeutic purposes that needs to be further clarified and developed.

L-PGDS deficiency accelerated the development of naturally occurring age-related osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder among the elderly. It is characterized by progressive cartilage degradation, synovial inflammation, subchondral bone remodeling and pain. Lipocalin prostaglandin D synthase (L-PGDS) is responsible for the biosynthesis of PGD2, which has been implicated in the regulation of inflammation and cartilage biology. This study aimed to evaluate the effect of L-PGDS deficiency on the development of naturally occurring age-related OA in mice. OA-like structural changes were assessed by histology, immunohistochemistry, and micro-computed tomography. Pain related behaviours were assessed using the von Frey and the open-field assays. L-PGDS deletion promoted cartilage degradation during aging, which was associated with enhanced expression of extracellular matrix degrading enzymes, matrix metalloprotease 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), and their breakdown products, C1,2C, VDIPEN and NITEG. Moreover, L-PGDS deletion enhanced subchondral bone changes, but had no effect on its angiogenesis. Additionally, L-PGDS deletion increased mechanical sensitivity and reduced spontaneous locomotor activity. Finally, we showed that the expression of L-PGDS was elevated in aged mice. Together, these findings indicate an important role for L-PGDS in naturally occurring age-related OA. They also suggest that L-PGDS may constitute a new efficient therapeutic target in OA.

Role of Lipocalin-Type Prostaglandin D Synthase in Experimental Osteoarthritis.

OBJECTIVE: Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the formation of prostaglandin D2 (PGD2 ), which has important roles in inflammation and cartilage metabolism. We undertook this study to investigate the role of L-PGDS in the pathogenesis of osteoarthritis (OA) using an experimental mouse model. METHODS: Experimental OA was induced in wild-type (WT) and L-PGDS-deficient (L-PGDS-/- ) mice (n = 10 per genotype) by destabilization of the medial meniscus (DMM). Cartilage degradation was evaluated by histology. The expression of matrix metalloproteinase 13 (MMP-13) and ADAMTS-5 was assessed by immunohistochemistry. Bone changes were determined by micro-computed tomography. Cartilage explants from L-PGDS-/- and WT mice (n = 6 per genotype) were treated with interleukin-1α (IL-1α) ex vivo in order to evaluate proteoglycan degradation. Moreover, the effect of intraarticular injection of a recombinant adeno-associated virus type 2/5 (rAAV2/5) encoding L-PGDS on OA progression was evaluated in WT mice (n = 9 per group). RESULTS: Compared to WT mice, L-PGDS-/- mice had exacerbated cartilage degradation and enhanced expression of MMP-13 and ADAMTS-5 (P < 0.05). Furthermore, L-PGDS-/- mice displayed increased synovitis and subchondral bone changes (P < 0.05). Cartilage explants from L-PGDS-/- mice showed enhanced proteoglycan degradation following treatment with IL-1α (P < 0.05). Intraarticular injection of rAAV2/5 encoding L-PGDS attenuated the severity of DMM-induced OA-like changes in WT mice (P < 0.05). The L-PGDS level was increased in OA tissues of WT mice (P < 0.05). CONCLUSION: Collectively, these findings suggest a protective role of L-PGDS in OA, and therefore enhancing levels of L-PGDS may constitute a promising therapeutic strategy.

In vivo protective effect of adipsin-deficiency on spontaneous knee osteoarthritis in aging mice.

The adipokine adipsin is an emerging mediator of human osteoarthritis (OA) progression. Here, we investigated its in vivo role in the development of spontaneous OA in aging mice. We compared articular knee joint morphology, histology in knee cartilage, synovial membrane, subchondral bone, meniscus, and anterior cruciate ligament (ACL); and chondrogenesis in the ACL from adipsin-deficient (Df-/-) and wild-type (Df+/+) 20-week- and 20-month-old mice. Serum levels of a panel of adipokines, inflammatory factors, and metalloproteases known to be implicated in OA were investigated. Data first revealed that the early manifestation of OA appeared in the ACL of 20-week-old mice, progressing to severe alterations in the 20 month-old wild-type mice. Further results demonstrated that adipsin-deficiency protected the articular tissues from spontaneous OA progression and triggered significantly higher serum levels of the adipokines adiponectin and FGF-21 while lowering levels of the inflammatory factor interleukin 6 (IL-6) in both young and old mice. This work further underlines the clinical relevance of adipsin as a novel therapeutic approach of human OA. Moreover, this study shows the potential beneficial effect of the adipokine FGF-21 against OA, and provides support for this factor to be a new biomarker and/or target of primary OA therapeutic avenues.

Empowering the immune fate of bone marrow mesenchymal stromal cells: gene and protein changes.

OBJECTIVE AND DESIGN: Bone marrow mesenchymal stromal cells (BM-MSCs) are referred as a promising immunotherapeutic cell product. New approaches using empowered MSCs should be developed as for the treatment or prevention of different immunological diseases. Such preconditioning by new licensing stimuli will empower the immune fate of BM-MSCs and, therefore, promote a better and more efficient biological. Here, our main goal was to establish the immunological profile of BM-MSCs following inflammatory priming and in particular their capacity to adjust their immune-related proteome and transcriptome. MATERIAL AND METHODS: To run this study, we have used BM-MSC cell cultures, a pro-inflammatory cytokine cocktail priming, flow cytometry analysis, qPCR and ELISA techniques. RESULTS: Different expression levels of several immunological mediators such as COX-1, COX-2, LIF, HGF, Gal-1, HO-1, IL-11, IL-8, IL-6 and TGF-ß were constitutively observed in BM-MSCs. Inflammation priming substantially but differentially modulated the gene and protein expression profiles of these mediators. Thus, expressions of COX-2, LIF, HGF, IL-11, IL-8 and IL-6 were highly increased/induced and those of COX-1, Gal-1, and TGF-ß were reduced. CONCLUSIONS: Collectively, we demonstrated that BM-MSCs are endowed with a specific and modular regulatory machinery which is potentially involved in immunomodulation. Moreover, BM-MSCs are highly sensitive to inflammation and respond to such signal by properly adjusting their gene and protein expression of regulatory factors. Using such preconditioning may empower the immune fate of MSCs and, therefore, enhance their value for cell-based immunotherapy.

Exacerbation of Aging-Associated and Instability-Induced Murine Osteoarthritis With Deletion of D Prostanoid Receptor 1, a Prostaglandin D2 Receptor.

OBJECTIVE: D prostanoid receptor 1 (DP1), a receptor for prostaglandin D2 , plays important roles in inflammation and cartilage metabolism. However, its role in the pathogenesis of osteoarthritis (OA) remains unknown. This study was undertaken to explore the roles of DP1 in the development of OA in murine models and to evaluate the efficacy of a DP1 selective agonist in the treatment of OA. METHODS: The development of aging-associated OA and destabilization of the medial meniscus (DMM)-induced OA was compared between DP1-deficient (DP1-/- ) and wild-type (WT) mice. The progression of OA was assessed by histology, immunohistochemistry, and micro-computed tomography. Cartilage explants from DP1-/- and WT mice were treated with interleukin-1α (IL-1α) ex vivo, to evaluate proteoglycan degradation. The effect of intraperitoneal administration of the DP1 selective agonist BW245C on OA progression was evaluated in WT mice. RESULTS: Compared to WT mice, DP1-/- mice had exacerbated cartilage degradation in both models of OA, and this was associated with increased expression of matrix metalloproteinase 13 and ADAMTS-5. In addition, DP1-/- mice demonstrated enhanced subchondral bone changes. Cartilage explants from DP1-/- mice showed enhanced proteoglycan degradation following treatment with IL-1α. Intraperitoneal injection of BW245C attenuated the severity of DMM-induced cartilage degradation and bony changes in WT mice. CONCLUSION: These findings indicate a critical role for DP1 signaling in OA pathogenesis. Modulation of the functions of DP1 may constitute a potential therapeutic target for the development of novel OA treatments.

Characterization of Mouse Models of Early Pancreatic Lesions Induced by Alcohol and Chronic Pancreatitis.

OBJECTIVE: We describe the first mouse model of pancreatic intraepithelial neoplasia (PanIN) lesions induced by alcohol in the presence and absence of chronic pancreatitis. METHODS: Pdx1-Cre;LSL-K-ras mice were exposed to Lieber-DeCarli alcohol diet for 6 weeks with cerulein injections. The PanIN lesions and markers of fibrosis, inflammation, histone deacetylation, epithelial-to-mesenchymal transition (EMT), and cancer stemness were measured by immunohistochemistry and Western. RESULTS: Exposure of Pdx1-Cre;LSL-K-ras mice to an alcohol diet significantly stimulated fibrosis and slightly but not significantly increased the level of PanIN lesions associated with an increase in tumor-promoting M2 macrophages. Importantly, the alcohol diet did not increase activation of stellate cells. Alcohol diet and cerulein injections resulted in synergistic and additive effects on PanIN lesion and M2 macrophage phenotype induction, respectively. Cerulein pancreatitis caused stellate cell activation, EMT, and cancer stemness in the pancreas. Pancreatitis caused histone deacetylation, which was promoted by the alcohol diet. Pancreatitis increased EMT and cancer stemness markers, which were not further affected by the alcohol diet. CONCLUSIONS: The results suggest that alcohol has independent effects on promotion of PDAC associated with fibrosis formed through a stellate cell-independent mechanism and that it further promotes early PDAC and M2 macrophage induction in the context of chronic pancreatitis.