Microenvironment-sensitive nanozymes for tissue regeneration

Bone, cartilage, and soft tissue regeneration is a complex spatiotemporal process recruiting a variety of cell types, whose activity and interplay must be precisely mediated for effective healing post-injury. Although extensive strides have been made in the understanding of the immune microenvironment processes governing bone, cartilage, and soft tissue regeneration, effective clinical translation of these mechanisms remains a challenge. Regulation of the immune microenvironment is increasingly becoming a favorable target for bone, cartilage, and soft tissue regeneration; therefore, an in-depth understanding of the communication between immune cells and functional tissue cells would be valuable. Herein, we review the regulatory role of the immune microenvironment in the promotion and maintenance of stem cell states in the context of bone, cartilage, and soft tissue repair and regeneration. We discuss the roles of various immune cell subsets in bone, cartilage, and soft tissue repair and regeneration processes and introduce novel strategies, for example, biomaterial-targeting of immune cell activity, aimed at regulating healing. Understanding the mechanisms of the crosstalk between the immune microenvironment and regeneration pathways may shed light on new therapeutic opportunities for enhancing bone, cartilage, and soft tissue regeneration through regulation of the immune microenvironment.

Background: Tissue regeneration is a crucial biological process with significant implications for developmental biology and regenerative medicine. Zebrafish (Danio rerio), known for their remarkable regenerative abilities, serve as an excellent model for studying tissue regeneration, particularly through the caudal fin regeneration model. Materials and Methods: β-Chitosan-TiO₂ nanoparticles were biosynthesized using plant extracts and microbial cultures. Characterization was performed using SEM, UV-Vis and FTIR. Zebrafish caudal fin regeneration was assessed post-amputation, with nanoparticle treatment in 16 μg/L and 32 μg/L. Regeneration was analyzed via microscopic observation and gene expression. Data were statistically analyzed using ANOVA, considering p < 0.05 significant. Results: β-Chitosan-TiO₂ nanoparticles exhibited uniform size, high stability, and strong biocompatibility. Zebrafish treated with β-Chitosan-TiO₂ nanoparticles showed significantly enhanced caudal fin regeneration compared to controls. Gene expression analysis supported upregulation of regenerative markers. Regenerated fin length measurements showed a dose-dependent improvement, with optimal effects at moderate nanoparticle concentrations. No significant toxicity or abnormalities were observed in treated zebrafish. Statistical analysis confirmed significant differences (p < 0.05) between control and treated groups, demonstrating β-Chitosan-TiO₂ NPs' potential in tissue regeneration. Conclusion: β-Chitosan-TiO₂ nanoparticles significantly enhanced zebrafish caudal fin regeneration by promoting cell proliferation and angiogenesis without toxicity. These findings highlight their potential as a biocompatible and eco-friendly nanomaterial for tissue regeneration applications.

Alginate and alginate composites for biomedical applications

Tissue regeneration and bone augmentation have slowly become mainstreams of periodontists’ daily practice. Biologic agents which attempt to promote soft and hard tissue repair as well as jump-start the healing process, especially in medically compromised individuals, are an integral part of clinical periodontics. Tissue engineering using biologic agents, such as enamel matrix derivative, platelet-derived growth factor, platelet-rich plasma or platelet-rich fibrin gel, bone morphogenetic protein, fibroblast growth factor, low dosage parathyroid hormone–related protein, or combination therapy, has recently become a focus of research and constant debate among clinicians, especially periodontists. These biologic agents have been tested and show promising results in promoting soft and hard tissue regeneration. The purpose of this special issue of Clinical Advances in Periodontics is to target this emerging field by inviting individuals who have expertise in this area to share their insights with our readers. All articles contain the most updated information and show how each biologic agent should be used in daily practice. Clinical examples of how each agent can be used in promoting tissue regeneration or bone augmentation are illustrated. In deciding which agent to use, it is important for clinicians to understand the mechanism of each biological modifier and the evidence that exists, as well as other factors such as cost/ benefit ratio, risk/benefit ratio, and long-term stability. Readers are encouraged to understand how these agents work, when to use them, and where they should be used, either alone or in combination, so they can make informed decisions in specific clinical situations. This special issue beginswith an introductionwith several focused questions such as: ‘‘What biologic agents are available to help promote periodontal regeneration and bone augmentation?’’; ‘‘How should clinicians decide which biologic agent(s) to use?’’; and ‘‘What are the approved uses for each agent?’’. The agents are then linked to their future perspectives in the second paper. In the following papers, the current available agents are introduced with clinical case illustrations as well as current evidence. The final paper demonstrates a combination approach using these agents. By reading this special issue of Clinical Advances in Periodontics, I hope readers gain a clear understanding of why, how, where, and when these biologic agents should be used in clinical practice to promote tissue andbone regeneration.n Hom-Lay Wang Guest Editor

Promising wound dressings can achieve rapid soft-tissue filling while refactoring the biochemical and biophysical microenvironment to recruit endogenous cells, facilitating tissue healing, integration, and regeneration. In this study, a tissue biomolecule-responsive hydrogel matrix, employing natural silk fibroin (SF) as a functional biopolymer and haemoglobin (Hb) as a peroxidase-like biocatalyst, was fabricated through cascade enzymatic crosslinking. The hydrogels possessed mechanical tunability and displayed adjustable gelation times. A tyrosine unit on SF stabilised the structure of Hb during the cascade oxidation process; thus, the immobilized Hb in SF hydrogels exhibited higher biocatalytic efficiency than the free enzyme system, which provided a continuously antioxidative system. The regulation of the dual enzyme ratio endowed the hydrogels with favourable biocompatibility, biodegradability, and adhesion strength. These multifunctional hydrogels provided a three-dimensional porous extracellular matrix-like microenvironment for promoting cell adhesion and proliferation. A rat model with a full-thickness skin defect revealed accelerated wound regeneration via collagen deposition, re-epithelialisation and revascularisation. Enzyme-loaded hydrogels are an attractive and high-safety biofilling material with the potential for wound healing, tissue regeneration, and haemostasis.

Extracellular vesicles (EVs)-based cell-free strategy has shown therapeutic potential in tissue regeneration. Due to their important roles in intercellular communications and their natural ability to shield cargos from degradation, EVs are also emerged as novel delivery vehicles for various bioactive molecules and drugs. Accumulating studies have revealed that EVs can be modified to enhance their efficacy and specificity for the treatment of many diseases. Engineered EVs are poised as the next generation of targeted delivery platform in the field of precision therapy. In this review, the unique properties of EVs are overviewed in terms of their biogenesis, contents, surface features and biological functions, and the recent advances in the strategies of engineered EVs construction are summarized. Additionally, we also discuss the potential applications of engineered EVs in targeted therapy of cancer and damaged tissues, and evaluate the opportunities and challenges for translating them into clinical practice.

<p class="abstract">Musculoskeletal medicine targets both cartilage regeneration and healing of soft tissues. Articular cartilage repair and regeneration is primarily considered to be due to its poor regenerative properties. Cartilage defects due to joint injury, aging, or osteoarthritis have low self-repair ability thus they are most often irreversible as well as being a major cause of joint pain and chronic disability. Unfortunately, current methods do not seamlessly restore hyaline cartilage and may lead to the formation of fibro- or continue hypertrophic cartilage. Deficiency of efficient modalities of therapy has invited research to combine stem cells, scaffold materials and environmental factors through tissue engineering. Articular cartilage tissue engineering aims to repair, regenerate, and hence improve the function of injured or diseased cartilage. This holds great potential and has evoked intense interest in improving cartilage therapy. Platelet-rich plasma (PRP) and/or stem cells may be influential for tissue repair as well as cartilage regenerative processes. A great promise to advance current cartilage therapies toward achieving a consistently successful modality has been held for addressing cartilage afflictions. The use of stem cells, novel biologically inspired scaffolds and, emerging nanotechnology may be the best way to reach this objective via tissue engineering. A current and emergent approach in the field of cartilage tissue engineering is explained in this review for specific application. In the future, the development of new strategies using stem cells seeded in scaffolds and the culture medium supplemented with growth factors could improve the quality of the newly formed cartilage<span lang="EN-IN">.</span></p>

IntroductionPeriodontitis is a chronic infectious disease and is the major cause of tooth loss and other oral health issues around the world. Periodontal tissue regeneration has therefore always been the ultimate goal of dentists and researchers. Existing fabrication methods mainly focused on a top–down tissue engineering strategy in which several drawbacks remain, including low throughput and limited diffusion properties resulting from a large sample size. Gelatin methacrylate (GelMA) is a kind of photocrosslinkable and biocompatible hydrogel, with the capacities of enabling cell encapsulation and regeneration of functional tissues. Here, we developed a novel method to fabricate GelMA/nanohydroxylapatite (nHA) microgel arrays using a photocrosslinkable strategy. The viability, proliferation, and osteogenic differentiation and in vivo osteogenesis of human periodontal ligament stem cells (hPDLSCs) encapsulated in microgels were evaluated. The results suggested that such microgels provide great potential for periodontal tissue repair and regeneration.MethodsMicrogel arrays were fabricated by blending different weight ratios of GelMA and nHA. hPDLSCs were encapsulated in GelMA/nHA microgels of various ratios for a systematic evaluation of cell viability, proliferation, and osteogenic differentiation. In vivo osteogenesis in nude mice was also studied.ResultsThe GelMA/nHA microgels exhibited appropriate microarchitecture, mechanical strength, and surface roughness, thus enabling cell adhesion and proliferation. Additionally, the GelMA/nHA microgels (10%/2% w/v) enhanced the osteogenic differentiation of hPDLSCs by elevating the expression levels of osteogenic biomarker genes, such as ALP, BSP, OCN, and RUNX2. In vivo ectopic transplantation results showed that GelMA/nHA microgels (10%/2% w/v) increased mineralized tissue formation with abundant vascularization, compared with the 1%, 3%, and the pure GelMA group.ConclusionThe GelMA/nHA microgels (10%/2% w/v) facilitated hPDLSCs viability, proliferation, and osteogenic differentiation in vitro and further promoted new bone formation in vivo, suggesting that the GelMA/nHA microgels (10%/2% w/v) provide great potential for periodontal tissue regeneration.

The aim of this study was the conception, production, material analysis and cytocompatibility analysis of a new collagen foam for medical applications. After the innovative production of various collagen sponges from bovine sources, the foams were analyzed ex vivo in terms of their structure (including pore size) and in vitro in terms of cytocompatibility according to EN ISO 10993-5/-12. In vitro, the collagen foams were compared with the established biomaterials cerabone and Jason membrane. Materials cerabone and Jason membrane. Collagen foams with different compositions were successfully produced from bovine sources. Ex vivo, the foams showed a stable and long-lasting primary structure quality with a bubble area of 1,000 to 2,000 μm2 In vitro, all foams showed sufficient cytocompatibility. Collagen sponges represent a promising material for hard and soft tissue regeneration. Future studies could focus on integrating and investigating different additives in the foams.

Purpose:Repair of distal phalanx amputation is widely studied and thought to occur through a regenerative process. Single-cell RNA sequencing (scRNA) has greatly improved the ability to characterize the cells that are involved in regeneration and abnormal healing of bone and soft tissue. Given the disorganized architecture of digit tip “regenerated” bone and its common progenitor cell to the pathologic process of heterotopic ossification, we hypothesized that digit tip repair after injury follows a program of aberrant wound repair rather than true regeneration of normal bone tissue.Methods:ScRNA data of mouse digit tip amputation (DTA) and embryonic limb development (EMB) was downloaded from NBCI GEO data base ascension number GSE135985. Additionally, scRNA data from heterotopic ossification (HO) was downloaded from GSE126060. HO, DTA, and EMB datasets were clustered individually. HO was merged and aligned with EMB and DT in separate analyses. Cells expressing the mesenchymal progenitor cell (MPC) marker Pdgfra, which we have shown to differentiate into HO and others have shown to differentiate into bone after DTA, were identified and analyzed in the HO-DTA and HO-EMB datasets. Monocle 2, Seurat 3.1.1, and Pearson correlation analysis were used for analysis and visualization. Immunostaining for markers present in DTA (ACAN, FBN-2, LTBP2) was performed on sections collected from a murine model of traumatic HO in Pdgfra-CreER;TdTomato mice induced with tamoxifen one-week prior to HO-forming injury.Results:MPC clusters from DTA and HO and from EMB and HO were aligned and projected using UMAP to compare transcription profiles. Across the timepoints, MPCs in the DTA and HO occupy similar regions of the UMAP plot, while MPCs in EMB and HO occupy largely independent regions, with only overlap at the latest time points. Using Monocle 2, cells were placed on a virtual timeline based on similarities in transcription. MPCs from DTA and HO meet at the same location on the trajectory following injury. In the EMB to HO comparison, cells only occupy similar regions of the trajectory at post-natal day 3 suggesting that bone is formed by different pathways. To understand correlations between time points, transcriptomes were also compared by Pearson analysis. MPCs following HO and DTA injuries occupy similar regions of the plot while MPCs in EMB and HO only occupy similar regions of the correlation plot at P3. Markers previously identified to be upregulated in MPCs in DTA were also upregulated in MPCs in HO by both scRNA and immunofluorescent histology.Conclusions:Cells responsible for bone repair following DTA show similar transcriptional profiles as MPCs in traumatic HO and dissimilar to MPCs in EMB. Furthermore, MPCs in both digit tip repair and HO follow trajectories that do not overlap with embryonic limb development. Thus, we demonstrate for the first time, that the process following DTA is not truly regeneration and follows programs of aberrant repair as seen in HO.

Adipose stem cells in tissue regeneration and repair: From bench to bedside

Composite scaffolds of bioactive glass and poly(lactide-co-glycolide) provide advantages over homogeneous scaffolds, yet their therapeutic potential can be improved by strategies that promote adhesion and present instructive cues to associated cells. Mesenchymal stem cell (MSC)-secreted extracellular matrix (ECM) enhances survival and function of associated cells. To synergize the benefits of an instructive ECM with composite scaffolds, we tested the capacity of ECM-coated composite scaffolds to promote cell persistence and resultant osteogenesis. Human MSCs cultured on ECM-coated scaffolds exhibited increased metabolic activity and decreased apoptosis compared to uncoated scaffolds. Additionally, MSCs on ECM-coated substrates in short-term culture secreted more proangiogenic factors while maintaining markers of osteogenic differentiation. Upon implantation, we detected improved survival of MSCs on ECM-coated scaffolds over 3 weeks. Histological evaluation revealed enhanced cellularization and osteogenic differentiation in ECM-coated scaffolds compared to controls. These findings demonstrate the promise of blending synthetic and natural ECMs and their potential in tissue regeneration.

Modulating degradation of sodium alginate/bioglass hydrogel for improving tissue infiltration and promoting wound healing

With the development of tissue engineering, the application of decellularized adipose matrix as scaffold material in tissue engineering has been intensively explored due to its wide source and excellent potential in tissue regeneration. Decellularized adipose matrix is a promising candidate for adipose tissue regeneration, while modification of decellularized adipose matrix scaffold can also allow it to transcend the limitations of adipose tissue source properties and applied to other tissue engineering fields, including cartilage and bone tissue engineering, neural tissue engineering, and skin tissue engineering. In this review, we summarized the development of the applications of decellularized adipose matrix in different tissue engineering and present future perspectives.Level of Evidence III This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

BackgroundThe fabrication of recombinant collagen and its prescribed variants has enormous potential in tissue regeneration, cell-matrix interaction investigations, and fundamental biochemical and biophysical studies of the extracellular matrix. Recombinant expression requires proline hydroxylation, a post-translational modification which is critical for imparting stability and structure. However, these modifications are not native to typical bacterial or yeast expression systems. Furthermore, detection of low levels of 4-hydroxyproline is challenging with respect to selectivity and sensitivity.ResultsWe have developed a new liquid chromatography-mass spectrometry (LC-MS) method to evaluate proline hydroxylation in recombinant collagen. This assay was tested in different Saccharomyces cerevisiae expression systems to evaluate the effect of gene ratio between prolyl-4-hydroxylase and collagen on the extent of hydroxylation. These systems used a human collagen III gene that was synthesized de novo from oligonucleotides. The LC-MS assay does not require derivatization, uses only picomoles of sample, and can measure proline hydroxylation levels in recombinant and native collagen ranging from approximately 0% to 40%. The hydroxylation values obtained by LC-MS are as accurate and as precise as those obtained with the conventional method of amino acid analysis.ConclusionsA facile, derivatization-free LC-MS method was developed that accurately determines the percentage of proline hydroxylation in different yeast expression systems. Using this assay, we determined that systems with a higher collagen-to-hydroxylase gene copy ratio yielded a lower percentage of hydroxylation, suggesting that a specifically balanced gene ratio is required to obtain higher hydroxylation levels.