In Vitro Mineralisation of Tissue-Engineered Cartilage Reduces Endothelial Cell Migration, Proliferation and Tube Formation.

Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with ß-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-ß for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor.

AIMS: Osteoarthritis (OA) is a common degenerative joint disease characterized by chronic inflammatory articular cartilage degradation. Long noncoding RNAs (lncRNAs) have been previously indicated to play an important role in inflammation-related diseases. Herein, the current study set out to explore the involvement of lncRNA H19 in OA. METHODS: Firstly, OA mouse models and interleukin (IL)-1ß-induced mouse chondrocytes were established. Expression patterns of IL-38 were determined in the synovial fluid and cartilage tissues from OA patients. Furthermore, the targeting relationship between lncRNA H19, tumour protein p53 (TP53), and IL-38 was determined by means of dual-luciferase reporter gene, chromatin immunoprecipitation, and RNA immunoprecipitation assays. Subsequent to gain- and loss-of-function assays, the levels of cartilage damage and proinflammatory factors were further detected using safranin O-fast green staining and enzyme-linked immunosorbent assay (ELISA) in vivo, respectively, while chondrocyte apoptosis was measured using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) in vitro. RESULTS: IL-38 was highly expressed in lentivirus vector-mediated OA mice. Meanwhile, injection of exogenous IL-38 to OA mice alleviated the cartilage damage, and reduced the levels of proinflammatory factors and chondrocyte apoptosis. TP53 was responsible for lncRNA H19-mediated upregulation of IL-38. Furthermore, it was found that the anti-inflammatory effects of IL-38 were achieved by its binding with the IL-36 receptor (IL-36R). Overexpression of H19 reduced the expression of inflammatory factors and chondrocyte apoptosis, which was abrogated by knockdown of IL-38 or TP53. CONCLUSION: Collectively, our findings evidenced that upregulation of lncRNA H19 attenuates inflammation and ameliorates cartilage damage and chondrocyte apoptosis in OA by upregulating TP53, IL-38, and by activating IL-36R.Cite this article: Bone Joint Res 2022;11(8):594-607.

Pharmacological Effects of Fasudil on Flap Survival in a Rodent Model.

BACKGROUND: Necrosis of the perforator flap is a critical problem. Fasudil, an inhibitor of Rho-associated coiled-coil containing kinase, has antiapoptosis activity and attenuates oxidative stress in many diseases. We characterized the effects of fasudil through intraperitoneal injection on perforator flap survival and identified its possible mechanism. METHODS AND MATERIALS: Rats were divided into a control group (without surgery), a flap group (only surgery), and a fasudil group (surgery plus fasudil). Perforator flaps were made on the backs of the rats. The expression of vascular endothelial growth factor, the protein kinase B (PKB/Akt), endothelial nitric oxide synthase, Bax, Bcl-2, Beclin-1, P62, and LC3 II/LC3 I was determined by Western blot at day 3 after surgery. Nitric oxide (NO) components, superoxide dismutase, and malondialdehyde were also measured at day 3. The survival rate and laser Doppler perfusion imaging were performed at day 7 after surgery. RESULT: The group with fasudil treatment exhibited the higher survival rates and angiogenesis levels. Fasudil also induced the activation of Akt/eNOS/NO pathway detected by the Western blot and NO expression kit. Furthermore, Western blot results showed fasudil-attenuated apoptosis through a raised Bcl-2/Bax rate and enhanced autophagy levels through raised beclin-1, decreased p62, and the elevated rate of LC3 II/LC3 I. Finally, fasudil increased superoxide dismutase and decreased malondialdehyde. CONCLUSIONS: In conclusion, fasudil treatment decreased necrosis of perforator flaps possibly by affecting the Akt/eNOS/NO pathway, attenuating apoptosis and activating autophagy.

Effects of bradykinin on the survival of multiterritory perforator flaps in rats.

BACKGROUND: Bradykinin, a vasoactive peptide, has many biological functions. For example, it accelerates angiogenesis. Thus, we studied the effects of bradykinin on the survival of perforator flaps. METHODS: Averagely, 50 male Sprague-Dawley rats were divided into control and bradykinin groups and underwent procedures to the multiterritory perforator flap. Areas of flap survival were tested 7 days later. Flap perfusion was evaluated by laser Doppler imaging. We assessed the extent of autophagy by determining LC3-II/I, Beclin 1, and p62. Flap angiogenesis was assessed by immunohistochemistry and H&E staining. We measured the level of vascular endothelial growth factor (VEGF) protein using western blot. We assessed oxidative stress by measuring the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) levels. The apoptotic index was also evaluated by western blot, and we determined nitric oxide (NO) production using an NO assay kit. RESULTS: The bradykinin group exhibited significantly larger areas of flap survival, higher blood supply, and more neovascularization. The bradykinin group also had higher SOD activity, higher VEGF expression and NO content, and reduced MDA compared to the control group. Rats treated with bradykinin also had lower levels of apoptosis and autophagy relative to the control group. CONCLUSION: Our results suggest that bradykinin promotes the survival of multiterritory perforator flaps by increasing angiogenesis, promoting the release of NO, suppressing apoptosis, reducing oxidative stress, and inhibiting autophagy.