Human Fibroblast-Derived Matrix Hydrogel Accelerates Regenerative Wound Remodeling Through the Interactions with Macrophages.

Herein, a novel extracellular matrix (ECM) hydrogel is proposed fabricated solely from decellularized, human fibroblast-derived matrix (FDM) toward advanced wound healing. This FDM-gel is physically very stable and viscoelastic, while preserving the natural ECM diversity and various bioactive factors. Subcutaneously transplanted FDM-gel provided a permissive environment for innate immune cells infiltration. Compared to collagen hydrogel, excellent wound healing indications of FDM-gel treated in the full-thickness wounds are noticed, particularly hair follicle formation via highly upregulated ß-catenin. Sequential analysis of the regenerated wound tissues disclosed that FDM-gel significantly alleviated pro-inflammatory cytokine and promoted M2-like macrophages, along with significantly elevated vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) level. A mechanistic study demonstrated that macrophages-FDM interactions through cell surface integrins α5ß1 and α1ß1 resulted in significant production of VEGF and bFGF, increased Akt phosphorylation, and upregulated matrix metalloproteinase-9 activity. Interestingly, blocking such interactions using specific inhibitors (ATN161 for α5ß1 and obtustatin for α1ß1) negatively affected those pro-healing growth factors secretion. Macrophages depletion animal model significantly attenuated the healing effect of FDM-gel. This study demonstrates that the FDM-gel is an excellent immunomodulatory material that is permissive for host cells infiltration, resorbable with time, and interactive with macrophages, where it thus enables regenerative matrix remodeling toward a complete wound healing.

Mesenchymal stem cell-derived secretomes-enriched alginate/ extracellular matrix hydrogel patch accelerates skin wound healing.

BACKGROUND: The secretomes of mesenchymal stem cells (MSCs) have great therapeutic potential and thereby their efficient delivery into the target site is of particular interest. Here, we propose a new strategy of hMSCs-derived secretomes delivery for advanced wound healing upon harnessing the working principle of extracellular matrix (ECM)-growth factors interaction in vivo. METHODS: We prepared an alginate hydrogel based wound patch, where it contains both human MSC-derived secretomes and ECM. The ECM was obtained from the decellularization of in vitro cultured human lung fibroblasts. The alginate solution was blended with ECM suspension, crosslinked, air-dried, then rehydrated with the secretomes contained in the concentrated conditioned media (CCM) as a highly saturated form of conditioned media (CM). We tested four different groups, with or without the ECM to investigate not only the role of ECM but the therapeutic effect of secretomes. RESULTS: The secretomes reserved many, diverse bioactive factors, such as VEGF, HGF, IGFBPs, IL-6, and IL-8. Alginate/ECM/CCM (AEC) patch could hold significantly larger amount of secretomes and release them longer than the other groups. Our AEC patch was the most effective in stimulating not only cell migration and proliferation but the collagen synthesis of dermal fibroblasts in vitro. Moreover, the AEC patch-treated full-thickness skin wounds disclosed significantly better wound healing indications: cell recruitment, neovascularization, epidermis thickness, keratinocyte migration, and mature collagen deposition, as assessed via histology (H&E, Herovici staining) and immunofluorescence, respectively. In particular, our AEC patch enabled a phenotype shift of myofibroblast into fibroblast over time and led to mature blood vessel formation at 14 day. CONCLUSIONS: We believe that ECM certainly contributed to generate a secretomes-enriched milieu via ECM-secretomes interactions and thereby such secretomes could be delivered more efficiently, exerting significant therapeutic impact either individually or collectively during wound healing process.

M2 Macrophage-Derived Concentrated Conditioned Media Significantly Improves Skin Wound Healing.

BACKGROUND: Macrophages, with many different phenotypes play a major role during wound healing process, secreting the cytokines crucial to angiogenesis, cell recruitment and ECM remodeling. Therefore, macrophage-derived cytokines may be attractive therapeutic resource for wound healing. METHODS: To obtain a conditioned media (CM) from macrophages, human monocyte THP-1 cells were seeded on TCP or human fibroblast-derived matrix (hFDM) and they were differentiated into M1 or M2 phenotype using distinct protocols. A combination of different substrates and macrophage phenotypes produced M1- and M2-CM or M1-hFDM- and M2-hFDM-CM, respectively. Proteome microarray determines the cytokine contents in those CMs. CMs-treated human dermal fibroblast (hDFB) was analyzed using collagen synthesis and wound scratch assay. Concentrated form of the CM (CCM), obtained by high-speed centrifugation, was administered to a murine full-thickness wound model using alginate patch, where alginate patch was incubated in the M2-CCM overnight at 4 °C before transplantation. On 14 day post-treatment, examination was carried out through H&E and Herovici staining. Keratinocyte and M2 macrophages were also evaluated via immunofluorescence staining. RESULTS: Cytokine analysis of CMs found CCL1, CCL5, and G-CSF, where CCL5 is more dominant. We found increased collagen synthesis and faster wound closure in hDFB treated with M2-CM. Full-thickness wounds treated by M2-hFDM-CCM containing alginate patch showed early wound closure, larger blood vessels, increased mature collagen deposition, enhanced keratinocyte maturation and more M2-macrophage population. CONCLUSION: Our study demonstrated therapeutic potential of the CM derived from M2 macrophages, where the cytokines in the CM may have played an active role for enhanced wound healing.

Nano-Sized Extracellular Matrix Particles Lead to Therapeutic Improvement for Cutaneous Wound and Hindlimb Ischemia.

Cell-derived matrix (CDM) has proven its therapeutic potential and been utilized as a promising resource in tissue regeneration. In this study, we prepared a human fibroblast-derived matrix (FDM) by decellularization of in vitro cultured cells and transformed the FDM into a nano-sized suspended formulation (sFDM) using ultrasonication. The sFDM was then homogeneously mixed with Pluronic F127 and hyaluronic acid (HA), to effectively administer sFDM into target sites. Both sFDM and sFDM containing hydrogel (PH/sFDM) were characterized via immunofluorescence, sol-gel transition, rheological analysis, and biochemical factors array. We found that PH/sFDM hydrogel has biocompatible, mechanically stable, injectable properties and can be easily administered into the external and internal target regions. sFDM itself holds diverse bioactive molecules. Interestingly, sFDM-containing serum-free media helped maintain the metabolic activity of endothelial cells significantly better than those in serum-free condition. PH/sFDM also promoted vascular endothelial growth factor (VEGF) secretion from monocytes in vitro. Moreover, when we evaluated therapeutic effects of PH/sFDM via the murine full-thickness skin wound model, regenerative potential of PH/sFDM was supported by epidermal thickness, significantly more neovessel formation, and enhanced mature collagen deposition. The hindlimb ischemia model also found some therapeutic improvements, as assessed by accelerated blood reperfusion and substantially diminished necrosis and fibrosis in the gastrocnemius and tibialis muscles. Together, based on sFDM holding a strong therapeutic potential, our engineered hydrogel (PH/sFDM) should be a promising candidate in tissue engineering and regenerative medicine.

Extracellular matrices derived from different cell sources and their effect on macrophage behavior and wound healing.

A decellularized extracellular matrix (dECM) is an excellent biomaterial in regenerative medicine, due to its biomimetic nature in targeting tissues and organs. In this study, we prepared cell-derived ECMs (CDM) derived from four different cell sources, characterized them individually, and found that intrinsic properties of each CDM were substantially different in terms of the fibrous matrix, total protein, and biochemical factors. Based on such information, we selected two ECM candidates, the human lung fibroblast derived matrix (hFDM) and the umbilical cord-blood mesenchymal stem cell derived matrix (UMDM) for the study of ECM-macrophage interactions in vitro and in vivo. In fact, UMDM was the richer in both total protein and angiogenic-related cytokines than any other CDM. When THP-1 cell-derived macrophages (M0) were seeded onto the UMDM or the hFDM, it showed a mixed cell morphology of macrophage phenotype and the macrophages (M0) preconditioned on UMDM presented more diverse cytokine release profiles. The treatment of conditioned medium obtained from CDM-seeded macrophages showed that UMDM could yield significantly advanced wound closure in 24 h via the human dermal fibroblast scratch model. To investigate the role of ECM on macrophage polarization in vivo, we prepared an ECM hydrogel, a mixture of each CDM and Pluronic F127/hyaluronan, and applied them onto a full-thickness mouse skin wound model for 2 weeks. The therapeutic efficacy as assessed via histology and immunofluorescence staining (α-SMA and CD206) revealed that the UMDM-treated group showed more effective wound healing compared to the other groups, as proven via the thinner epidermal layer, significant recovery of skin appendage, better neovascularization, and higher recruitment of myofibroblasts and larger number of macrophages (M2) at 7 days. The difference between UMDM and hFDM was marginal. Taken together, among the CDMs, UMDM and hFDM are promising resources of ECM, showing a great potential for wound healing. Although the mechanism is not fully understood, bioactive innate factors in UMDM may contribute individually and/or collectively to advance wound healing.

An injectable, self-assembled multicellular microsphere with the incorporation of fibroblast-derived extracellular matrix for therapeutic angiogenesis.

Decellularized human lung fibroblast-derived matrix (hFDM) has demonstrated its excellent proangiogenic capability. In this study, we propose a self-assembled, injectable multicellular microspheres containing human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cell (MSCs), collagen hydrogel (Col), and hFDM toward therapeutic angiogenesis. Those multicellular microspheres are spontaneously formed by the mixtures of cell and hydrogel after being dropped on the parafilm for several hours. The size of microspheres can be manipulated via adjusting the initial volume of droplets and the culture period. The cells in the microspheres are highly viable. Multicellular microspheres show good capability of cell migration on 2D culture plate and also exhibit active cell sprouting in 3D environment (Col) forming capillary-like structures. We also find that multiple angiogenic-related factors are significantly upregulated with the multicellular microspheres prepared via Col and hFDM (Col/hFDM) than those prepared using Col alone or single cells (harvested from cocultured HUVECs/MSCs in monolayer). For therapeutic efficacy evaluation, three different groups of single cells, Col and Col/hFDM microspheres are injected to a hindlimb ischemic model, respectively, along with PBS injection as a control group. It is notable that Col/hFDM microspheres significantly improve the blood reperfusion and greatly attenuate the fibrosis level of the ischemic regions. In addition, Col/hFDM microspheres show higher cell engraftment level than that of the other groups. The incorporation of transplanted cells with host vasculature is detectable only with the treatment of Col/hFDM. Current results suggest that hFDM plays an important role in the multicellular microspheres for angiogenic cellular functions in vitro as well as in vivo. Taken together, our injectable multicellular microspheres (Col/hFDM) offer a very promising platform for cell delivery and tissue regenerative applications.