Journal of Tissue Engineering and Regenerative Medicine最新文献

Human mesenchymal stem cells (hMSCs) are multipotent cells that differentiate into adipocytes, chondrocytes, and osteoblasts. Owing to their differentiation potential, hMSCs are among the cells most frequently used for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, which necessitates in vitro expansion. Conventionally, this is often performed on rigid surfaces such as tissue culture plates (TCPs). However, during in vitro expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, rendering them unsuitable for clinical use. Although multiple approaches have been attempted to maintain hMSC stemness during prolonged expansion, finding a suitable culture system remains an unmet need. Recently, a few research groups have shown that hMSCs maintain their stemness over long passages when cultured on soft substrates. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. Furthermore, it has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay replicative senescence in hMSCs during long-term expansion on TCPs. However, the mechanism by which chromatin condensation/decondensation influences nuclear morphology and DNA damage, which are strongly related to the onset of senescence, remains unknown. To answer this question, we cultured hMSCs for long duration in the presence of epigenetic modifiers, histone acetyltransferase inhibitor (HATi), which promotes chromatin condensation by preventing histone acetylation, and histone deacetylase inhibitor (HDACi), which promotes chromatin decondensation, and investigated their effects on various nuclear markers related to senescence. We found that consistent acetylation causes severe nuclear abnormalities, whereas chromatin condensation by deacetylation helps to safeguard the nucleus from damage caused by in vitro expansion.

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed in vitro, which included cell migration, viability, and apoptosis. In vivo, a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&E) were used to observe femoral trabeculae. Our in vitro results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts in vitro. Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. In vivo, increased bone volume per tissue volume (BV/TV) (p = 0.031), trabecular thickness (Tb.Th) (p = 0.020), and decreased separation (Tb.Sp) (p = 0.040) were observed in SNFH rats treated with EC-exos. H&E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.

Saethre-Chotzen syndrome (SCS) is one of the most prevalent craniosynostosis, caused by a loss-of-function mutation in the TWIST-1 gene, with current treatment options relying on major invasive transcranial surgery. TWIST-1 haploinsufficient osteogenic progenitor cells exhibit increased osteogenic differentiation potential due to an upregulation of the transmembrane tyrosine kinase receptor, C-ROS-1, a TWIST-1 target gene known to promote bone formation. The present study assessed the efficacy of suppressing C-ROS-1 activity using a known chemical inhibitor to C-ROS-1, crizotinib, to halt premature coronal suture fusion in a preclinical mouse model of SCS. Crizotinib (1 μM, 2 μM, or 4 μM) was administered locally over the calvaria of Twist-1^del/+ heterozygous mice prior to coronal suture fusion using either a nonresorbable collagen sponge (quick drug release) or a resorbable sodium carboxymethylcellulose microdisk (slow sustained release). Coronal suture fusion rates and bone parameters were determined by μCT imaging and histomorphometric analysis of calvaria postcoronal suture fusion. Results demonstrated a dose-dependent increase in the efficacy of crizotinib to maintain coronal suture patency, with no adverse effects to brain, kidney, liver, and spleen tissue, or blood cell parameters. Moreover, crizotinib delivered on microdisks resulted in a greater efficacy at a lower concentration to reduce bone formation at the coronal suture sites compared to sponges. However, the bone inhibitory effects were found to be diminished by over time following cessation of treatment. Our findings lay the foundation for the development of a pharmacological nonsurgical, targeted approach to temporarily maintain open coronal sutures in SCS patients. This study could potentially be used to develop similar therapeutic strategies to treat different syndromic craniosynostosis conditions caused by known genetic mutations.

While acute wounds typically progress through the phases of wound healing, chronic wounds often stall in the inflammatory phase due to elevated levels of matrix metalloproteinases (MMPs) and proinflammatory cytokines. Dysregulated expression of MMPs can result in the breakdown of extracellular matrix (ECM) formed during the wound healing process, resulting in stalled wounds. Native collagen-based wound dressings offer a potential wound management option to sequester excess MMPs and support cellular interactions that allow wound progression through the natural healing process. Herein, we utilized commercially available ECM matrices, two derived from porcine small intestinal submucosa (PCMP, 2 layers; PCMP-XT, 5 layers) and one derived from propria submucosa (ovine forestomach matrix, OFM, 1 layer), to demonstrate the impact of processing methodologies (e.g., layering and crosslinking) on functional characteristics needed for the management of chronic wounds. Grafts were evaluated for structural composition using scanning electron microscopy and histology, ability to reduce MMPs using fluorometric assays, and durability in an in vitro degradation chronic wound model. Both intact (nondegraded) and partially degraded grafts were assessed for their ability to serve as a functional cell scaffold using primary human fibroblasts. Grafts differed in matrix substructure and composition. While all grafts demonstrated attenuation of MMP activity, PCMP and PCMP-XT showed larger reductions of MMP levels. OFM rapidly degraded in the in vitro degradation model (<3 hours), while PCMP and PCMP-XT were significantly more durable (>7 days). The ability of PCMP and PCMP-XT to serve as scaffolds for cellular attachment was not impacted by degradation in vitro. Three ECM grafts with varying structural and functional characteristics exhibited differential durability when degraded in a simulated chronic wound model. Those that withstood rapid degradation maintained their ability to function as a scaffold to support attachment and proliferation of fibroblasts, a cell type important for wound healing.

Introduction. The decellularized human amniotic membrane (dHAM) emerges as a viable 3D scaffold for organ repair and replacement using a tissue engineering strategy. Glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) can increase the biomechanical properties of dHAM. However, the crosslinking process is associated with biochemical changes and residual toxic materials, dampening the biocompatibility of the dHAM. From a histologic point of view, each side of the amniotic membrane is biologically different. While the dHAM basement membrane side is rich in growth factors, the stromal side of the dHAM contains more connective tissue matrix (e.g., collagen fibers) which supports its biomechanical properties. Biocompatibility and biomechanical properties are two important challenges in the field of materials science. In this study, for the first time, the stromal and basement membrane side are cross-linked with GTA and EDC, respectively, to optimize the biocompatibility of the treated dHAM while sparing the GTA-mediated biomechanical improvements. Methods. Crosslinking was carried out on dHAM in three groups: EDC, GTA and bilateral treatment with EDC&GTA. Mechanical resistance, degradability, and crosslinking measurements were performed on treated dHAM. The viability of mesenchymal stem cells (MSCs) on the scaffolds was evaluated by the MTT assay. The expression levels of surface markers and images of the MSCs were thoroughly studied. Results. The results obtained showed that bilateral treatment of dHAM with EDC and GTA increased mechanical resistance. Similarly, the evaluation of surface markers revealed that bilaterally treated dHAM sustains the stemness and viability of MSCs at a level equal to that achieved with EDC alone. The SEM images indicated that the MSCs maintained adhesion on EDC&GTA-cross-linked dHAM. Conclusion. The current study explores a pioneering treatment of dHAM, a material long recognized for its regenerative properties, in a novel context. This research delves into the utilization of dHAM cross-linked with EDC&GTA, demonstrating its optimized efficacy in tissue engineering. The enhanced crosslinking technique significantly alters the membrane’s properties, amplifying its durability and therapeutic potential. In this novel bilateral treatment strategy (EDC and GTA), improving mechanical properties by GTA on the stromal surface and maintaining the biocompatibility of EDC on the side of the basement membrane of dHAM had been attained together. By investigating the handling and impact of this cross-linked membrane, this study unveils a new approach in leveraging a well-known material through an innovative process, revolutionizing its application in wound care.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease. Patients can present with inflammatory nodules, abscesses up to fistulas, or sinus tracts in intertriginous body parts. Occlusion of the sebaceous gland unit leads to its rupture, with a subsequent exuberant immune response. Given there is still no causative therapy, to better understand HS and develop novel therapeutic concepts, research activities in the HS field are constantly growing. Primary skin cells, blood cells, and ex vivo explant cultures from HS patients have been previously used as HS cell culture models. In vitro reconstituted epidermal models are established to study inflammatory dermatoses, such as psoriasis or atopic dermatitis. For HS, the exploration of epidermis models would be an excellent addition, e.g., biomarkers or barrier function in testing new topic treatment options. We therefore established a stratified in vitro HS epidermis model based on primary cells from HS lesions. After isolating keratinocytes from lesional skin, we cultured them submerged in a transwell system. To induce differentiation, we then lifted them to the air-liquid interface. Immunohistochemical staining demonstrated that our HS-epidermis model meets the expected differentiation pattern. In addition, we detected the secretion of the inflammatory cytokines interleukin-1β and TNF-α.

In rheumatoid arthritis, dysregulated cytokine signaling has been implicated as a primary factor in chronic inflammation. Many antirheumatic and biological therapies are used to suppress joint inflammation, but despite these advances, effectiveness is not universal, and delivery is often at high doses, which can predispose patients to significant off-target effects. During chronic inflammation, the inappropriate regulation of signaling factors by macrophages accelerates the progression of disease by driving an imbalance of inflammatory cytokines, making macrophages an ideal cellular target. To develop a macrophage-based therapy to treat chronic inflammation, we engineered a novel induced pluripotent stem cell (iPSC)-derived macrophage capable of delivering soluble TNF receptor 1 (sTNFR1), an anti-inflammatory biologic inhibitor of tumor necrosis factor alpha (TNF-α), in an autoregulated manner in response to TNF-α. Murine iPSCs were differentiated into macrophages (iMACs) over a 17-day optimized protocol with continued successful differentiation confirmed at key timepoints. Varying inflammatory and immunomodulatory stimuli demonstrated traditional macrophage function and phenotypes. In response to TNF-α, therapeutic iMACs produced high levels of sTNFR1 in an autoregulated manner, which inhibited inflammatory signaling. This self-regulating iMAC system demonstrated the potential for macrophage-based drug delivery as a novel therapeutic approach for a variety of chronic inflammatory diseases.

Chronic tympanic membrane (TM) perforation increases patient susceptibility to infection, hearing loss, and other side effects. Current clinical treatment, surgical grafting, can result in detrimental side effects including nerve damage, dizziness, or hearing loss. Therefore, it is essential to develop novel therapeutic procedures that can induce or accelerate healing in minimally or noninvasive approaches. Cell-free therapies have safety advantages over stem cells and are logistically favorable for clinical use. The regenerative potential by mesenchymal stem cell-conditioned media (CM) has been promising. In this study, poly(lactic-co-glycolic acid) (PLGA) microspheres with CM encapsulated have been developed as a cell-free alternative regenerative treatment for TM perforation. The results suggest that the PLGA microspheres were capable of encapsulating and releasing CM for up to 21 days. The in vitro scratch wound proliferation assays showed increased wound healing ability of CM-loaded microspheres. In vivo guinea pig models treated with CM drops and CM-loaded microspheres using a thermoresponsive gel carrier demonstrated potential for wound healing in TM perforation. These studies provide a basis for further examination of the delivery of stem cell CM and investigation of time-dependent wound healing, long-term ototoxicity, and hearing restoration.

Therapeutic electric fields (EFs) are applied to the epidermis to accelerate the healing of chronic epidermal wounds and promote skin transplantation. While research has emphasized understanding the role of EFs in polarizing the migration of superficial epidermal cells, there are no reports describing the effect of EFs on polarization of the underlying vasculature. We explored the effects of EFs on the growth of endothelial sprouts, precursors to functional blood vessels. We discovered that DC EFs of the same magnitude near wounded epidermis polarize initiation, growth, and turning of endothelial sprouts toward the anode. While EFs polarize sprouts, they do not change the frequency of primary sprout or branch formation. Unidirectional electrical pulses also polarize sprouts based on their time-averaged EF magnitude. Sprout polarization occurs antiparallel to the direction of electrically driven water flow (electro-osmosis) and is consistent with the direction of sprout polarization induced by pressure-driven flow. These results support the role of EFs in controlling the direction of neovascularization during the healing of soft tissues and tissue engineering.

Polycaprolactone (PCL) is a promising material for the fabrication of alternatives to autologous grafts used in coronary bypass surgery. PCL biodegrades over time, allowing cells to infiltrate the polymeric matrix, replacing the biodegrading graft, and creating a fully functional vessel constituted of autologous tissue. However, the high hydrophobicity of PCL is associated with poor cell affinity. Surface modification of PCL can increase this cell affinity, making PCL an improved scaffold material for acellular vascular grafts. In this study, the surface of PCL films was modified by hydrolysis, aminolysis, and the combination thereof to introduce carboxyl, hydroxyl, and amino groups on the surface. Only the hydrolyzed films exhibited a significant increase in their hydrophilicity, although further testing showed that all aminolysis conditions had amino groups on the surface. Furthermore, in vitro experiments with human umbilical endothelial cells (HUVECs) were performed to assess changes in cell affinity for PCL due to the surface treatments. PCL treated with sodium hydroxide (NaOH), a hydrolysis reaction, showed a significant increase in endothelial cell adhesion after 24 hours with a significant increase in cell survival after 72 hours. Thus, NaOH treatment improves the biocompatibility and endothelialization of PCL, creating a competent candidate for artificial, acellular, biodegradable vascular grafts.