Tissue engineering and regenerative medicine最新文献

Background: Soft tissue integration (STI) around dental implant abutments is a prerequisite to prevent bacterial invasion and achieve successful dental implant rehabilitation. However, peri-implant STI is a major challenge after dental abutment placement due to alterations in the immune microenvironment upon surgical dental implant installation.

Methods: Based on known immunomodulatory effects of zinc, we herein deposited zinc/chitosan/gelatin (Zn/CS/Gel) coatings onto titanium substrates to study their effect on macrophages. First, we exposed macrophages to cell culture media containing different zinc ion (Zn²⁺) concentrations. Next, we explored the immunomodulatory effect of Zn/CS/Gel coatings prepared via facile electrophoretic deposition (EPD).

Results: We found that Zn²⁺ effectively altered the secretome by reducing the secretion of pro-inflammatory and enhancing pro-regenerative cytokine secretion, particularly at a Zn²⁺ supplementation of approximately 37.5 μM. Zn/CS/Gel coatings released Zn²⁺ in a concentration range which effectively stimulated pro-regenerative macrophage polarization as demonstrated by M2 macrophage polarization. Additionally, the impact of these Zn²⁺-exposed macrophages on gingival fibroblasts incubated in conditioned medium showed stimulated adhesion, proliferation, and collagen secretion.

Conclusion: Our promising results suggest that controlled release of Zn²⁺ from Zn/CS/Gel coatings could be applied to immunomodulate peri-implant STI, and to enhance dental implant survival.

Background: Polylactic acid (PLA) is extensively used in the medical and cosmetic industries for skin regeneration and as a dermal filler due to its biocompatibility and biodegradability. However, the effectiveness of PLA as a cosmetic filler is limited by its slow degradation rate and poor cell attachment properties. Recent studies have focused on enhancing the performance of PLA by combining it with other materials. This study aimed to evaluate the performance of carboxymethyl cellulose (CMC), known for its high biocompatibility, in comparison with the widely used hyaluronic acid (HA).

Methods: Two types of PLA-based particles, HA-PLA and CMC-PLA were synthesized by combining PLA with HA and CMC, respectively. After characterizing the particles, we evaluated cell adhesion and viability using human dermal fibroblasts and analyzed gene and protein expression related to cell attachment and angiogenic paracrine factors.

Results: The CMC-PLA particles maintained a more uniform size distribution than the HA-PLA particles and exhibited superior cell adhesion properties. Cells attached on the CMC-PLA particles showed enhanced secretion of angiogenic paracrine factors, suggesting a potential improvement in therapeutic efficacy.

Conclusion: CMC-PLA particles demonstrated superior cell adhesion and secretion capabilities compared with HA-PLA particles, indicating their potential for application in skin regeneration and tissue recovery. Further research, including in vivo studies, is required to fully explore and validate the therapeutic potential of CMC-PLA particles.

Background: Diabetes mellitus with nonalcoholic fatty liver disease (DM-NAFLD) represents a complex metabolic syndrome with significant clinical challenges. This study explores the therapeutic potential and underlying mechanisms of umbilical cord-derived mesenchymal stem cells (UCMSCs)-derived extracellular vesicles (EVs) in DM-NAFLD.

Methods: UCMSCs-EVs were isolated and characterized. DM-NAFLD mouse model was developed through high-energy diet and streptozotocin injection. Additionally, primary mouse hepatocytes were exposed to high glucose to simulate cellular conditions. Hepatic tissue damage, body weight changes, lipid levels, glucose and insulin homeostasis, and hepatic lipid accumulation were evaluated. The interaction between UCMSCs-EVs and hepatocytes was assessed, focusing on the localization and function of circ-Tulp4. The study also investigated the expression of circularRNA TUB-like protein 4 (circ-Tulp4), heterogeneous nuclear ribonucleoprotein C (HNRNPC), abhydrolase domain containing 6 (ABHD6), cleaved Caspase-1, NLR family pyrin domain containing 3 (NLRP3) and cleaved N-terminal gasdermin D (GSDMD-N). The binding of circ-Tulp4 to lysine demethylase 6B (KDM6B) and the subsequent epigenetic regulation of ABHD6 by H3K27me3 were analyzed.

Results: Circ-Tulp4 was reduced, while HNRNPC and ABHD6 were elevated in DM-NAFLD models. UCMSCs-EVs attenuated hepatic steatosis and inhibited the NLRP3/cleaved Caspase-1/GSDMD-N pathway. EVs delivered circ-Tulp4 into hepatocytes, thereby restoring circ-Tulp4 expression. Elevated circ-Tulp4 enhanced the recruitment of H3K27me3 to the HNRNPC promoter through interaction with KDM6B, thus suppressing HNRNPC and ABHD6. Overexpression of HNRNPC or ABHD6 counteracted the protective effects of UCMSCs-EVs, exacerbating pyroptosis and hepatic steatosis in DM-NAFLD.

Conclusion: UCMSCs-EVs deliver circ-Tulp4 into hepatocytes, where circ-Tulp4 inhibits the HNRNPC/ABHD6 axis, thereby reducing pyroptosis and alleviating DM-NAFLD. These findings provide a novel therapeutic avenue for targeting DM-NAFLD through modulation of cell pyroptosis.

Background: Tendinopathy is a chronic tendon disease. Mesenchymal stem cells (MSCs), known for their anti-inflammatory properties, may lose effectiveness with extensive culturing. Previous research introduced "small umbilical cord-derived fast proliferating cells" (smumf cells), isolated using a novel minimal cube explant method. These cells maintained their MSC characteristics through long-term culture. Thus, the purpose of the present study was to assess the anti-inflammatory effects of late-passage smumf cells at P10 on tenocytes derived from degenerative rotator cuff tears in a tendinopathic environment.

Methods: The mRNA expression with respect to aging of MSCs and secretion of growth factors (GFs) by smumf cells at P10 were measured. mRNA and protein synthesis in tenocytes with respect to the tenocyte phenotype, inflammatory cytokines, and matrix- degradation enzymes were measured. The inflammatory signal pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) in tenocytes were also investigated. The proliferative response of degenerative tenocytes to co-culture with smumf cells over 7 days in varying IL-1β induced tendinopathic environments was investigated.

Results: smumf cells at P10 showed no signs of aging compared to those at P3. smumf cells at P10, secreting 2,043 pg/ml of hepatocyte growth factor (HGF), showed a 1.88-fold (p = .002) increase in HGF secretion in a tendinopathic environment. Degenerative tenocytes co-cultured with smumf cells showed significantly increased protein expression levels of collagen type I (Col I) and the Col I/III ratio by 1.46-fold (p < .001) and 1.66-fold (p < .001), respectively. The smumf cells at P10 reduced both mRNA and protein expression levels of matrix metalloproteinases-1, -2, -3, -8, -9, and -13 in tenocytes and attenuated NF-κB (phosphorylated IκBα/IκBα and phosphorylated p65/p65) and MAPK (phosphorylated p38/p38 and phosphorylated JNK/JNK) pathways activated by IL-1β. Removal of IL-1β from the co-culture accelerated the growth of tenocytes by 1.42-fold (p < .001). Removal of IL-1β accelerated tenocyte growth in co-cultures.

Conculsion: Late-passage smumf cells exert anti-inflammatory effects on tenocytes derived from degenerative rotator cuff tears under a tendinopathic environment, primarily through the secretion of growth factors (GFs).

Background: Injectable platelet-rich fibrin (iPRF), a liquid form of PRF that is prepared from peripheral blood without anticoagulants, promotes tissue wound healing and regeneration. The present study focused on iPRF-like bone marrow aspirate concentrate (iBMAC) prepared without anticoagulant, and the regenerative potential of iPRF and iBMAC was compared in vitro.

Methods: iPRF and iBMAC were prepared from the same New Zealand white rabbits. The cytocompatibility and regenerative potential of each concentrate were evaluated using primary rabbit gingival fibroblasts and osteoblasts.

Results: Both gingival fibroblasts and osteoblasts treated with each concentrate exhibited excellent cell viability. Interestingly, compared to cells treated with iPRF, cells treated with iBMAC demonstrated significantly greater migration potential. Furthermore, higher mRNA levels of transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and collagen I (COL1) were observed in gingival fibroblasts treated with iBMAC than in those treated with iPRF. Compared with osteoblasts treated with iPRF, osteoblasts treated with iBMAC exhibited greater differentiation potential, as indicated by increased osteocalcin (OCN) expression and mineralization capability.

Conclusion: The results of the in vitro study suggest that, compared with iPRF, iBMAC may promote wound healing and bone regeneration more effectively. However, further preclinical and clinical studies are needed to confirm the regenerative potential of iBMAC in the body.

Background: Hepatocytes are an attractive cell source in hepatic tissue engineering because they are the primary cells of the liver, maintaining liver homeostasis through their intrinsic function. Due to the increasing demand for liver donors, a wide range of methods are being studied to obtain functionally active hepatocytes. iPSCs are one of the alternative cell sources, which shows great promise as a tool for generating hepatocytes.

Methods: This study determined whether factors associated with iPSCs contributed to variation in hepatocyte-like cells derived from iPSCs. The factors of concern for the iPSCs included the culture system, the source of iPSCs, and cell seeding density for initiating the differentiation.

Results: Our results found iPSC-dependent variances among differentiated hepatocyte-like cells. The matrix used in culturing iPSCs significantly impacts cell morphologies, characteristics, and the expression of pluripotent genes, such as OCT4 and SOX2, varied in iPSCs derived from different sources. These characteristics, in turn, play a consequential role in determining the functional activity of the iPSC-derived hepatocyte-like cells. In addition, cell seeding density was observed to be an essential factor for the efficient generation of iPSC-derived hepatocyte-like cells, with 2- 4 × 10 cells/cm of seeding density resulting in good morphology and functionality.

Conclusion: This study provides the baseline of effective differentiation protocols for iPSC-derived hepatocyte-like cells with the appropriate conditions, including cell culture media, iPSC source, and the seeding density of iPSCs.

Background: Priming strategies that improve the functionality of MSCs may be required to address issues limiting successful clinical translation of MSC therapies. For conditions requiring high trophic support such as brain and spinal cord injuries, priming MSCs to produce higher levels of trophic factors may be instrumental to facilitate translation of current MSC therapies. We developed and tested a novel molecular priming paradigm using docosahexaenoic acid (DHA) to prime adipose tissue-derived mesenchymal stromal cells (ASCs) to enhance the secretome neuroregulatory potential.

Methods: Comprehensive dose-response and time-course assays were carried to determine an optimal priming protocol. Secretome total protein measurements were taken in association with cell viability, density and morphometric assessments. Cell identity and differentiation capacity were studied by flow cytometry and lineage-specific markers. Cell growth was assessed by trypan-blue exclusion and senescence was probed over time using SA-β-gal, morphometry and gene expression. Secretomes were tested for their ability to support differentiation and neurite outgrowth of human neural progenitor cells (hNPCs). Neuroregulatory proteins in the secretome were identified using multiplex membrane arrays.

Results: Priming with 40 µM DHA for 72 h significantly enhanced the biosynthetic capacity of ASCs, producing a secretome with higher protein levels and increased metabolic viability. DHA priming enhanced ASCs adipogenic differentiation and adapted their responses to replicative senescence induction. Furthermore, priming increased concentrations of neurotrophic factors in the secretome promoting neurite outgrowth and modulating the differentiation of hNPCs.

Conclusions: These results provide proof-of-concept evidence that DHA priming is a viable strategy to improve the neuroregulatory profile of ASCs.

Background: Periodontitis is a severe chronic inflammatory disease, whose traditional systemic antimicrobial therapy faces great limitations. In-situ gels provide an effective solution as an emerging local drug delivery system.

Methods: In this study, the novel thermosensitive poloxamer/carbopol in-situ gels loaded with 20 μmol/L quercetin for the treatment of periodontitis were prepared by cold method. Thirteen batches of in-situ gels based on two independent factors (X₁: poloxamer 407 and X₂: carbopol 934P) were designed and optimized by the statistical method of central composite design (CCD). The transparency, pH, injectability, viscosity, gelation temperature, gelation time, elasticity modulus, degradation rate and in-vitro drug release studies of the batches were evaluated, and the percentage of drug release in the first hour, the time required for 90% drug release, gelation temperature, and gelation time were selected as dependent variables.

Results: These two independent factors significantly affected the four dependent variables (p < 0.05). The optimization result displayed that the optimized concentration of poloxamer 407 was 20.84% (w/v), and carbopol 934P was 0.5% (w/v). The optimized formulation showed a clear appearance (++), acceptable injectability (Pass), viscosity(151,798 mPa s), gelation temperature (36 °C), gelation time (213 s), preferable cell viability and cell proliferation, conformed to first-order release kinetics, and had a significant antibacterial effect.

Conclusions: The article demonstrates the great potential of the quercetin in-situ gel as an effective treatment for periodontitis.

Background: Factor H and membrane inhibitor of reactive lysis (CD59) are key regulators of complement activation. Mesenchymal stem cells (MSCs) secrete Factor H and express CD59 to protect themselves from complement-mediated damage. Severe hypoxia found to decrease the survival chances of MSCs after transplantation; however, little is known about the impact of severe hypoxia on modulating the complement system activity and its effect on MSCs survival. Our study seeks to explore the effect of severe hypoxia on modulating the complement cascade in MSCs.

Methods: Human adipose tissue-derived MSCs (hAD-MSCs) were cultured under severe hypoxia using 400 μM Cobalt Chloride (CoCl2) for 48 h. The protein expressions of survival marker; Phosphoinositide 3-kinases (PI3K), and pro-apoptotic marker; Caspase-3 were assessed using western blotting. The level of complement system related factors; Factor H, CD59, C3b, iC3b, C5b, C9, and the complement membrane attack complex (MAC) were analyzed using Elisa assays, western blotting, and immunocytochemistry.

Results: Our results showed for the first time that severe hypoxia can significantly impair Factor H secretion and CD59 expression in MSCs. This has been associated with upregulation of MAC complex and increased level of cell lysis and apoptosis marked by downregulation of PI3K and upregulation of Annexin v and Caspase-3.

Conclusion: The loss of Factor H and CD59 in hypoxic MSCs can initiate their lysis and apoptosis mediated by activating MAC complex. Preserving the level of Factor H and CD59 in MSCs has significant clinical implication to increase their retention rate in hypoxic conditions and prolong their survival.

Background: In vitro cell culture is crucial for studying human diseases and development. Compared to traditional monolayer cultures, 3D culturing with organoids offers significant advantages by more accurately replicating natural tissues' structural and functional features. This advancement enhances disease modeling, drug testing, and regenerative medicine applications. Organoids, derived from stem cells, mimic tissue physiology in a more relevant manner. Despite their promise, the clinical use of regenerative medicine currently needs to be improved by reproducibility, scalability, and maturation issues.

Methods: This article overviews recent organoid research, focusing on their types, sources, 3D culturing methods, and applications in regenerative medicine. A literature review of "organoid" and "regenerative medicine" in PubMed/MEDLINE highlighted relevant studies published over the past decade, emphasizing human-sourced organoids and their regenerative benefits, as well as the availability of free full-text articles. The review uses descriptive data, including tables and text, to illustrate the challenges and potential of organoids in regenerative medicine.

Results: The transition from 2D to 3D models, particularly organoids, has significantly advanced in vitro research. This review covers a decade of progress in various organoid types-such as liver, cholangiocyte, intestinal, pancreatic, cardiac, brain, thymus, and mammary organoids-and their 3D culture methods and applications. It addresses critical issues of maturity, scalability, and reproducibility and underscores the need for standardization and improved production techniques to facilitate broader clinical applications in regenerative medicine.

Conclusions: Successful therapy requires increased scalability and standardization. Organoids have enormous potential in biological research, notwithstanding obstacles.