Suppression of SOCS3 expression in macrophage cells: Potential application in diabetic wound healing.

Impaired polarization of M1 to M2 macrophages has been reported in diabetic wounds. We aimed to improve this polarization by down-regulation of expression of the "Suppressor of Cytokine Signaling 3" (SOCS3) gene in macrophages. Two oligodeoxynucleotide (ASO) sequences were designed against SOC3 mRNA and were loaded to mannosylated-polyethyleneimine (Man-PEI). The optimum N/P ratio for Man-PEI-ASO was determined to be 8 based on loading efficiency, particle size, zeta potential, cellular uptake and cytotoxicity assay. pH stability of ASO in Man-PEI-ASO and its protection from DNase I was confirmed. After in vitro treatment of macrophages with Man-PEI-ASO, SOCS3 was downregulated, SOCS1 upregulated, and SOCS1/SOCS3 ratio increased. Also, expressions of macrophage markers of M2 (IL-10, Arg1, CD206) increased and those of M1 (IL-1ß, NOS2, CD68) decreased, and secretion of pro-inflammatory cytokines (TNF-α and IL-1ß) decreased while that of anti-inflammatory cytokine IL-4 increased. All suggested a polarization into M2 phenotype. Finally, the Man-PEI-ASO was loaded in hydrogel and applied to a diabetic wound model in mice. It improved the healing to the level observed in non-diabetic wounds. We show that using antisense sequences against SOC3 mRNA, macrophage polarization could be directed into the M2 phenotype and healing of diabetic wound could be highly improved.

Coculture of adipose-derived mesenchymal stem cells/macrophages on decellularized placental sponge promotes differentiation into the osteogenic lineage.

BACKGROUND: Several factors like three-dimensional microstructure, growth factors, cytokines, cell-cell communication, and coculture with functional cells can affect the stem cells behavior and differentiation. The purpose of this study was to investigate the potential of decellularized placental sponge as adipose-derived mesenchymal stem cells (AD-MSCs) and macrophage coculture systems, and guiding the osteogenic differentiation of stem cells. METHODS: The decellularized placental sponge (DPS) was fabricated, and its mechanical characteristics were evaluated using degradation assay, swelling rate, and pore size determination. Its structure was also investigated using hematoxylin and eosin staining and scanning electron microscopy. Mouse peritoneal macrophages and AD-MSCs were isolated and characterized. The differentiation potential of AD-MSCs co-cultured with macrophages was evaluated by RT-qPCR of osteogenic genes on the surface of DPS. The in vivo biocompatibility of DPS was determined by subcutaneous implantation of scaffold and histological evaluations of the implanted site. RESULTS: The DPS had 67% porosity with an average pore size of 238 µm. The in vitro degradation assay showed around 25% weight loss during 30 days in PBS. The swelling rate was around 50% during 72 h. The coculture of AD-MSCs/macrophages on the DPS showed a significant upregulation of four differentiation osteogenic lineage genes in AD-MSCs on days 14 and 21 and a significantly higher mineralization rate than the groups without DPS. Subcutaneous implantation of DPS showed in vivo biocompatibility of scaffold during 28 days follow-up. CONCLUSIONS: Our findings suggest the decellularized placental sponge as an excellent bone substitute providing a naturally derived matrix substrate with biostructure close to the natural bone that guided differentiation of stem cells toward bone cells and a promising coculture substrate for crosstalk of macrophage and mesenchymal stem cells in vitro.

Stem cell-mediated angiogenesis in skin tissue engineering and wound healing.

The timely management of skin wounds has been an unmet clinical need for centuries. While there have been several attempts to accelerate wound healing and reduce the cost of hospitalisation and the healthcare burden, there remains a lack of efficient and effective wound healing approaches. In this regard, stem cell-based therapies have garnered an outstanding position for the treatment of both acute and chronic skin wounds. Stem cells of different origins (e.g., embryo-derived stem cells) have been utilised for managing cutaneous lesions; specifically, mesenchymal stem cells (MSCs) isolated from foetal (umbilical cord) and adult (bone marrow) tissues paved the way to more satisfactory outcomes. Since angiogenesis plays a critical role in all four stages of normal wound healing, recent therapeutic approaches have focused on utilising stem cells for inducing neovascularisation. In fact, stem cells can promote angiogenesis via either differentiation into endothelial lineages or secreting pro-angiogenic exosomes. Furthermore, particular conditions (e.g., hypoxic environments) can be applied in order to boost the pro-angiogenic capability of stem cells before transplantation. For tissue engineering and regenerative medicine applications, stem cells can be combined with specific types of pro-angiogenic biocompatible materials (e.g., bioactive glasses) to enhance the neovascularisation process and subsequently accelerate wound healing. As such, this review article summarises such efforts emphasising the bright future that is conceivable when using pro-angiogenic stem cells for treating acute and chronic skin wounds.

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.

Tenocyte-imprinted substrate: a topography-based inducer for tenogenic differentiation in adipose tissue-derived mesenchymal stem cells.

Tendon tissue engineering based on stem cell differentiation has attracted a great deal of attention in recent years. Previous studies have examined the effect of cell-imprinted polydimethylsiloxane (PDMS) substrate on induction differentiation in stem cells. In this study, we used tenocyte morphology as a positive mold to create a tenocyte-imprinted substrate on PDMS. The morphology and topography of this tenocyte replica on PDMS was evaluated with scanning electron microscopy (SEM) and atomic force microscopy. The tenogenic differentiation induction capacity of the tenocyte replica in adipose tissue-derived mesenchymal stem cells (ADSCs) was then investigated and compared with other groups, including tissue replica (which was produced similarly to the tenocyte replica and was evaluated by SEM), decellularized tendon, and bone morphogenic protein (BMP)-12, as other potential inducers. This comparison gives us an estimate of the ability of tenocyte-imprinted PDMS (called cell replica in the present study) to induce differentiation compared to other inducers. For this reason, ADSCs were divided into five groups, including control, cell replica, tissue replica, decellularized tendon and BMP-12. ADSCs were seeded on each group separately and investigated by the real-time reverse transcription polymerase chain reaction (RT-PCR) technique after seven and 14 days. Our results showed that in spite of the higher effect of the growth factor on tenogenic differentiation, the cell replica can also induce tenocyte marker expression (scleraxis and tenomodulin) in ADSCs. Moreover, the tenogenic differentiation induction capacity of the cell replica was greater than tissue replica. Immunocytochemistry analysis revealed that ADSCs seeding on the cell replica for 14 days led to scleraxis and tenomodulin expression at the protein level. In addition, immunohistochemistry indicated that contrary to the promising results in vitro, there was little difference between ADSCs cultured on tenocyte-imprinted PDMS and untreated ADSCs. The results of such studies could lead to the production of inexpensive cell culture plates or biomaterials that can induce differentiation in stem cells without growth factors or other supplements.

Oxygen-Releasing Scaffolds for Accelerated Bone Regeneration.

Hypoxia, the result of disrupted vasculature, can be categorized in the main limiting factors for fracture healing. A lack of oxygen can cause cell apoptosis, tissue necrosis, and late tissue healing. Remedying hypoxia by supplying additional oxygen will majorly accelerate bone healing. In this study, biphasic calcium phosphate (BCP) scaffolds were fabricated by robocasting, an additive manufacturing technique. Then, calcium peroxide (CPO) particles, as an oxygen-releasing agent, were coated on the BCP scaffolds. Segmental radial defects with the size of 15 mm were created in rabbits. Uncoated and CPO-coated BCP scaffolds were implanted in the defects. The empty (control) group received no implantation. Repairing of the bone was investigated via X-ray, histological analysis, and biomechanical tests at 3 and 6 months postoperatively, with immunohistochemical examinations at 6 months after operation. According to the radiological observations, formation of new bone was augmented at the interface between the implant and host bone and internal pores of CPO-coated BCP scaffolds compared to uncoated scaffolds. Histomorphometry analysis represented that the amount of newly formed bone in the CPO-coated scaffold was nearly two times higher than the uncoated one. Immunofluorescence staining revealed that osteogenic markers, osteonectin and octeocalcin, were overexpressed in the defects treated with the coated scaffolds at 6 months of postsurgery, demonstrating higher osteogenic differentiation and bone mineralization compared to the uncoated scaffold group. Furthermore, the coated scaffolds had superior biomechanical properties as in the case of 3 months after surgery, the maximal flexural force of the coated scaffolds reached to 134 N, while it was 92 N for uncoated scaffolds. The results could assure a boosted ability of bone repair for CPO-coated BCP scaffolds implanted in the segmental defect of rabbit radius because of oxygen-releasing coating, and this system of oxygen-generating coating/scaffold might be a potential for accelerated repairing of bone defects.

Decellularization and preservation of human skin: A platform for tissue engineering and reconstructive surgery.

A matrix derived from natural tissue functions as a highly biocompatible and versatile scaffold for tissue engineering applications. It can act as a supportive construct that provides a niche for colonization by host cells. In this work, we describe a cost-effective, reliable and reproducible protocol for decellularization and preservation of human skin as a potential soft tissue replacement. The decellularized human skin is achieved using purely chemical agents without any enzymatic steps. The suitability of the proposed method for the preservation of the extracellular matrix (ECM) structure and its main components and integrity were evaluated using histological and immunohistochemical analysis. Cryopreservation and final sterility were conducted using programmable freeze-drying and gamma irradiation. The architecture, basement membrane and 3D structure of ECM can be successfully preserved after decellularization. Our protocol was found to be appropriate to maintain key proteins such as collagen type I, III, IV and laminin in the structure of final scaffold. This protocol offers a novel platform for the preparation of a dermal substitute for potential clinical applications. STATEMENT OF SIGNIFICANCE: Clinical application of naturally-based scaffolds for verity of health problems obliges development of a reproducible and effective technology that does not change structural and compositional material properties during scaffold preparation and preservation. Lack of an effective protocol for the production of biological products using decellularization method is still remaining. This effort is directing to solve this challenge in order to accomplish the off-the -shelf availability of decellularized dermal scaffold in market for clinical application.

3D Protein-Based Bilayer Artificial Skin for the Guided Scarless Healing of Third-Degree Burn Wounds in Vivo.

Severe burn injuries can lead to delays in healing and devastating scar formation. Attempts have been made to develop a suitable skin substitute for the scarless healing of such skin wounds. Currently, there is no effective strategy for completely scarless healing after the thermal injuries. In our recent work, we fabricated and evaluated a 3D protein-based artificial skin made from decellularized human amniotic membrane (AM) and electrospun nanofibrous silk fibroin (ESF) in vitro. We also characterized both biophysical and cell culture investigation to establish in vitro performance of the developed bilayer scaffolds. In this report, we evaluate the appropriate utility of this fabricated bilayered artificial skin in vivo with particular emphasis on healing and scar formation due to the biochemical and biomechanical complexity of the skin. For this work, AM and AM/ESF membranes alone or seeded with adipose-tissue-derived mesenchymal stem cells (AT-MSCs) are implanted on full-thickness burn wounds in mice. The healing efficacy and scar formation are evaluated at 7, 14, and 28 days post-implantation in vivo. Our data reveal that ESF accelerates the wound-healing process through the early recruitment of inflammatory cells such as macrophages into the defective site as well as the up-regulation of angiogenic factors from the AT-MSCs and the facilitation of the remodeling phase. In vivo application of the prepared AM/ESF membrane seeded with the AT-MSCs reduces significantly the post-burn scars. The in vivo data suggest that the potential applications of the AM/ESF bilayered artificial skin may be considered a clinical translational product with stem cells to guide the scarless healing of severe burn injuries.

Osteogenic potential of stem cells-seeded bioactive nanocomposite scaffolds: A comparative study between human mesenchymal stem cells derived from bone, umbilical cord Wharton's jelly, and adipose tissue.

Bone regeneration is considered as an unmet clinical need, the aim of this study is to investigate the osteogenic potential of three different mesenchymal stem cells (MSCs) derived from human bone marrow (BM-MSCs), umbilical cord Wharton's jelly (UC-MSCs), and adipose (AD-MSCs) seeded on a recently developed nanocomposite scaffold (bioactive glass/gelatin) implanted in rat animal models with critical size calvarial defects. In this study, after isolation, culture, and characterization, the MSCs were expanded and seeded on the scaffolds for in vitro and in vivo studies. The adhesion, proliferation, and viability of the cells on the scaffolds evaluated in vitro, showed that the scaffolds were biocompatible for further examinations. In order to evaluate the scaffolds in vivo, rat animal models with critical size calvarial defects were randomly categorized in four groups and treated with the scaffolds. The animals were sacrificed at the time points of 4 and 12 weeks of post-implantation, bone healing process were investigated. The histological and immunohistological observations showed (p < 0.01) higher osteogenesis capacity in the group treated with BM-MSCs/scaffolds compared to the other groups. However, the formation of new angiogenesis was evidently higher in the defects filled with UC-MSCs/scaffolds. This preliminary study provides promising data for further clinical trials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 61-72, 2018.

Strontium- and cobalt-substituted bioactive glasses seeded with human umbilical cord perivascular cells to promote bone regeneration via enhanced osteogenic and angiogenic activities.

Designing and developing new biomaterials to accelerate bone healing are currently under progress. In this study, we attempted to promote osteogenesis using strontium- and cobalt-substituted bioactive glasses (BGs) seeded with human umbilical cord perivascular cells (HUCPVCs) in a critical size defect in the distal femur of rabbit animal model. The BG particles were successfully synthesized in the form of granules using the melt-derived route. After being isolated, HUCPVCs were expanded and then characterized to use during in vitro and in vivo procedures. The in vitro effects of the synthesized glasses on the isolated HUCPVCs as well as on cell lines SaOS-2 (selected for screening the osteogenetic potential) and HUVEC (selected for screening the angiogenic potential) were assessed by analyzing cytotoxicity, cell attachment, bone-like nodule formation, and real time PCR. The results of in vitro tests indicated cytocompatibility of the synthesized BG particles. For in vivo study, the HUCPVCs-seeded BGs were implanted into the animal's body. Radiographic imaging, histology and immunohistology staining were performed on the harvested specimens at 4 and 12weeks post-surgery. The in vivo evaluation of the samples showed that all the cell/glass constructs accelerated bone healing process in comparison with blank controls. The best in vitro and in vivo results were associated to the BGs containing both strontium and cobalt ions. This group of bioactive glasses is able to promote both osteogenesis and angiogenesis and can therefore be highly suitable for the development of advanced functional bone substitutes. STATEMENT OF SIGNIFICANCE: Bone regeneration is considered as an unmet clinical need. The most recent researches focused on incorporation of strontium (Sr2+) and cobalt (Co2+) ions into bioactive glasses structure. Strontium is an alkaline earth metal which is currently used in the treatment of osteoporosis. Also, cobalt is considered as another promising element in the bone regeneration field that may induce hypoxia-mediated angiogenesis. In this study, the osteogenic potential of the strontium- and cobalt-substituted bioactive glasses in granule form seeded with human umbilical cord perivascular cells (HUCPVCs) was evaluated in vitro and in vivo. Indeed, the main goal of this study was to improve the osteogenenic and angiogenic properties of bioactive glasses through the incorporation of strontium and cobalt ions in the glass composition. Although some researches have been conducted on this subject, the influence of the simultaneous use of strontium and cobalt ions on the improvement of bone healing in vivo has been not yet well understood and, therefore, deserves further investigation.