Designing and synthesis of injectable hydrogel based on carboxymethyl cellulose/carboxymethyl chitosan containing QK peptide for femoral head osteonecrosis healing.

Femoral head necrosis is a debilitating disorder that typically caused by impaired blood supply to the hip joint. In this study, a novel injectable hydrogel based on Oxidized Carboxymethyl Cellulose (OCMC)-Carboxymethyl Chitosan (CMCS) polymers containing an angiogenesis stimulator peptide (QK) with a non-toxic crosslinking interaction (Schiff based reaction) was synthesized to enhance angiogenesis following femoral head necrosis in an animal model. The physicochemical features of fabricated injectable hydrogel were analyzed by FTIR, swelling and degradation rate, rheometry, and peptide release. Also, the safety and efficacy were evaluated following an in vitro hydrogel injection study and an avascular necrosis (AVN) animal model. According to the results, the hydrogel exhibited an appropriate swelling ratio and water uptake (>90 %, 24 h) as well as a suitable degradation rate over 21 days accompanied by a continuous peptide release. Also, data showed that hydrogels containing QK peptide boosted the proliferation, differentiation, angiogenesis, and osteogenic potential of both Bone Marrow mesenchymal Stem Cells (BM-MSCs) and human umbilical vein endothelial cells (HUVECs) (****p < 0.0001 and ***p < 0.001, respectively). Furthermore, molecular and histological evaluations significantly demonstrated the overexpression of Runx2, Osteocalcin, Collagen I, VEGF and CD34 genes (**p < 0.01 and ***p < 0.001, respectively), and also femoral head necrosis was effectively prohibited, and more blood vessels were detected in defect area by OCMC-CMCS hydrogel containing QK peptide (bone trabeculae >9000, ***p < 0.001). In conclusion, the findings demonstrate that OCMC-CMCS-QK injectable hydrogel could be considered as an impressive therapeutic construct for femoral head AVN healing.

Fabrication and characterization of bilayer scaffolds made of decellularized dermis/nanofibrous collagen for healing of full-thickness wounds.

Skin tissue engineering has progressed from simple wound dressings to biocompatible materials with desired physico-chemical properties that can deliver regenerative biomolecules. This study describes using a novel biomimetic hybrid scaffold of decellularized dermis/collagen fibers that can continuously deliver stromal cell-derived factor-1 alpha (SDF-1α) for skin regeneration. In diabetic rat models, the idea that sustained SDF-1α infusion could increase the recruitment of CXCR4-positive cells at the injury site and improve wound regeneration was investigated. The morphology of the scaffold, its biocompatibility, and the kinetics of SDF-1 release were all assessed. SDF-1α was successfully incorporated into collagen nanofibers, resulting in a 200-h continuous release profile. The microscopic observations exhibited that cells are attached and proliferated on proposed scaffolds. As evaluated by in vivo study and histological examination, fabricated scaffold with SDF-1α release capacity exhibited a remarkably more robust ability to accelerate wound regeneration than the control group. Besides, the SDF-1α-loaded scaffold demonstrated functional effects on the proliferation and recruitment of CD31 and CXCR4-positive cells in the wound bed. Additionally, no adverse effects such as hyperplasia or scarring were found during the treatment period. It may be concluded that the fabricated hybrid scaffold based on natural polymer opens up a new option for topical administration of bioactive molecules. We believe the SDF-1α-loaded hybrid scaffold has promise for skin tissue engineering.

Transplantation of decellularised human amniotic membranes seeded with mesenchymal stem cell-educated macrophages into animal models.

The reconstruction of chronic skin wounds remains a public health challenge in dermatology. Precisely controlling and monitoring the wound-healing process should result in enhanced outcomes for the patient. Cell-based therapies have shown great potential in medicine due to their immunomodulatory and healing properties. Herein, we produced activated macrophages by treating circulating monocytes with mesenchymal stem cell (MSC) supernatant. We also demonstrated the critical role of activated macrophages transplantation using amniotic membranes in accelerating wound healing in an animal wound model. The activated macrophages not only exhibited immunomodulatory cytokines like transforming growth factorß (TGFß) and interleukin 10 (and IL10) secretion but also showed attachment and proliferation ability on the amniotic membrane scaffold. Moreover, MSCs supernatant-treated cells also displayed significant ARG1, CD206, and IL 10 genes expression. Inspired by the in vitro results, we examined the in vivo therapeutic efficacy of the activated macrophage transplantation using an acellular amniotic membrane carrier in a full-thickness cutaneous wound model. The wound healing rate was significant in the group treated with macrophages generated via mesenchymal cell therapy seeded human amniotic membrane. There was less scarring in the wound sites after placing cell-scaffold constructs in the wound sites in the animal models. Overall, macrophages stimulated with mesenchymal cells' supernatant exhibited improved healing processes in incisional wounds by decreasing the inflammatory phase, increasing angiogenesis, and reducing scar tissue development.

In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens.

BACKGROUND: L7/L12 is a protective antigen conserved in main Brucella pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all Brucella pathogens. MATERIALS AND METHODS: The purpose of the current study was to in silico design a L7/L12-SOmp2b fusion protein and in vitro production of the chimera. Two possible fusion forms, L7/L12-SOmp2b and SOmp2b-L7/L12, were subjected to in silico modeling and analysis. Cloning and expression of the fusion protein has been done in the pET28a vector and Escherichia coli Bl21 (DE3), respectively. RESULTS: Analysis and validation of the fusion proteins three-dimensional models showed that both models are in the range of native proteins. However, L7/L12-SOmp2b structure was more valid than the SOmp2b-L7/L12 model and subjected to in vitro production. The major histocompatibility complex II (MHC-II) epitope mapping using Immune Epitope DataBase indicated that the model contained good MHC-II binders. The L7/L12-Omp2b coding sequence was cloned in pET28a vector. The fusion was successfully expressed in E. coli BL21 by induction with isopropyl-ß-d-thiogalactopyranoside. The rL7/L12-SOmp2b was purified with Ni-NTA column. The yield of the purified rL7/L12-SOmp2b was estimated by Bradford method to be 240 µg/ml of the culture. Western blot analysis revealed a specific reactivity with purified rL7/L12-SOmp2b produced in E. coli cells and showed the expression in the prokaryotic system. CONCLUSIONS: Our data indicates that L7/L12-SOmp2b fusion protein has a potential to induce both B- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.

Comparison of potential protection conferred by three immunization strategies (protein/protein, DNA/DNA, and DNA/protein) against Brucella infection using Omp2b in BALB/c Mice.

In the present study, immunogenicity and protective efficacy of the Brucella outer membrane protein 2b (Omp2b) was evaluated in BALB/c mice using Protein/Protein, DNA/DNA and DNA/Protein vaccine strategies. Immunization of mice with three vaccine regimens elicited a strong specific IgG response (higher IgG2a titers over IgG1 titers) and provided Th1-oriented immune response. Vaccination of BALB/c mice with the DNA/Pro regimen induced higher levels of IFN-γ/IL-2 and conferred more protection levels against B. melitenisis and B. abortus challenge than did the protein or DNA alone. In conclusion, Omp2b is able to stimulate specific immune responses and to confer cross protection against B. melitensis and B. abortus infection. Therefore, it could be introduced as a new potential candidate for the development of a subunit vaccine against Brucella infection.