Endogenous Repair Research Articles

Multifunctional polyphenol-based silk hydrogel alleviates oxidative stress and enhances endogenous regeneration of osteochondral defects

In osteochondral defects, oxidative stress caused by elevated levels of reactive oxygen species (ROS) can disrupt the normal endogenous repair process. In this study, a multifunctional hydrogel composed of silk fibroin (SF) and tannic acid (TA), the FDA-approved ingredients, was developed to alleviate oxidative stress and enhance osteochondral regeneration. In this proposed hydrogel, SF first interacts with TA to form a hydrogen-bonded supramolecular structure, which is subsequently enzymatically crosslinked to form a stable hydrogel. Furthermore, TA had multiple phenolic hydroxyl groups that formed interactions with the therapeutic molecule E7 peptide for controlled drug delivery. In vitro investigations showed that SF-TA and SF-TA-E7 hydrogels exhibited a multitude of biological effects including scavenging of ROS, maintaining cell viability, and promoting the proliferation of bone marrow mesenchymal stem cells (BMSCs) against oxidative stress. The proteomic analysis indicated that SF-TA and SF-TA-E7 hydrogels suppressed oxidative stress, which in turn improved cell proliferation in multiple proliferation and apoptosis-related pathways. In rabbit osteochondral defect model, SF-TA and SF-TA-E7 hydrogels promoted enhanced regeneration of both cartilage and subchondral bone as compared to hydrogel without TA incorporation. These findings indicated that the multifunctional SF-TA hydrogel provided a microenvironment suitable for the endogenous regeneration of osteochondral defects.

Iron promotes neurological function recovery in mice with ischemic stroke through endogenous repair mechanisms

The endogenous repair mechanisms play an important role in the recovery of nerve function after stroke, such as gliosis, synaptic plasticity, remyelination and nerve regeneration. Iron is the most abundant trace metal element in the brain and plays a crucial role in the maintenance of normal cerebral function. It is an important coenzyme factor in the process of cell metabolism, DNA synthesis, purine catabolism and neurotransmitter synthesis and decomposition. However, it is unclear what role iron plays in the long-term recovery of neurological function after stroke. In this study, we first observed that changes in iron metabolism occurred during neurological function recovery in the mice with distal middle cerebral artery occlusion (dMCAO). Our data showed that plasticity changes due to endogenous repair mechanisms resulted in improvements in cerebral cortex function. These changes involved gliosis, synaptic function reconstruction, remyelination, and activation of neural stem cells. In order to examine the potential role of iron, we synthesized liposomal-encapsulated deferoxamine (DFO) nanoparticles to further explore the effect and the mechanism of iron on the recovery of neurological function in dMCAO mice. Our results showed that liposome-DFO decreased iron deposition and reversed plasticity changes in cerebral cortex function after stroke, which delayed neurological function recovery. This experiment shows that the increasing iron level promotes endogenous repair in ischemic stroke. Our finding reveals the change regularity of iron and emphasizes the beneficial role of iron in the recovery process of neurological function, which provides an important basis for the prevention and/or treatment of ischemia-reperfusion and recovery after stroke.

Follistatin promotes LIN28B-mediated supporting cell reprogramming and hair cell regeneration in the murine cochlea

Hair cell (HC) loss within the inner ear cochlea is a leading cause for deafness in humans. Before the onset of hearing, immature supporting cells (SCs) in neonatal mice have some limited capacity for HC regeneration. Here, we show that in organoid culture, transient activation of the progenitor-specific RNA binding protein LIN28B and Activin antagonist follistatin (FST) enhances regenerative competence of maturing/mature cochlear SCs by reprogramming them into progenitor-like cells. Transcriptome profiling and mechanistic studies reveal that LIN28B drives SC reprogramming, while FST is required to counterbalance hyperactivation of transforming growth factor–β–type signaling by LIN28B. Last, we show that LIN28B and FST coactivation enhances spontaneous cochlear HC regeneration in neonatal mice and that LIN28B may be part of an endogenous repair mechanism that primes SCs for HC regeneration. These findings indicate that SC dedifferentiation is critical for HC regeneration and identify LIN28B and FST as main regulators.

Platelet-Derived Growth Factor-Functionalized Scaffolds for the Recruitment of Synovial Mesenchymal Stem Cells for Osteochondral Repair.

Cartilage regeneration is still a challenge for clinicians because of avascularity, denervation, load-bearing, synovial movement, and the paucity of endogenous repair cells. We constructed a multilayered osteochondral bionic scaffold and examined its repair capacity using a rabbit osteochondral defect model. The cartilage phase and interface layer of the scaffold were prepared by freeze-drying, whereas the bone phase of the scaffold was prepared by high-temperature sintering. The three-phase osteochondral bionic scaffold was formed by joining the hydroxyapatite (HAp) and silk fibroin (SF) scaffolds using the repeated freeze-thaw method. Different groups of scaffolds were implanted into the rabbit osteochondral defect model, and their repair capacities were assessed using imaging and histological analyses. The cartilage phase and the interface layer of the scaffold had a pore size of 110.13 ± 29.38 and 96.53 ± 33.72 μm, respectively. All generated scaffolds exhibited a honeycomb porous structure. The polydopamine- (PDA-) modified scaffold released platelet-derived growth factor (PDGF) for 4 weeks continuously, reaching a cumulative release of 71.74 ± 5.38%. Synovial mesenchymal stem cells (SMSCs) adhered well to all scaffolds, but demonstrated the strongest proliferation ability in the HSPP (HAp-Silk-PDA-PDGF) group. Following scaffold-induced chondrogenic differentiation, SMSCs produced much chondrocyte extracellular matrix (ECM). In in vivo experiments, the HSPP group exhibited a significantly higher gross tissue morphology score and achieved cartilage regeneration at an earlier stage and a significantly better repair process compared with the other groups (P < 0.05). Histological analysis revealed that the new cartilage tissue in the experimental group had a better shape and almost filled the defect area, whereas the scaffold was nearly completely degraded. The new cartilage was effectively fused with the surrounding normal cartilage, and a substantial amount of chondrocyte ECM was formed. The SF/HAp three-layer osteochondral bionic scaffold exhibited favorable pore size, porosity, and drug sustained-release properties. It demonstrated good biocompatibility in vitro and encouraging repair effect at osteochondral defect site in vivo, thereby expected to enabling the repair and regeneration of osteochondral damage.

Differences in the expression profiles of lncRNAs and mRNAs in partially injured anterior cruciate ligament and medial collateral ligament of rabbits.

Long noncoding RNAs (lncRNAs), as a novel regulatory factor, are considered to play a vital role in various biological processes and diseases. However, the overall expression profile and biological functions of lncRNAs in the partially injured anterior cruciate ligament (ACL) and medial collateral ligament (MCL) have not been clearly explored. Partially injured models of ACL and MCL were established in 3-month-old healthy male New Zealand white rabbits. Expression of lncRNAs and mRNAs in the ligament tissue was detected by high-throughput sequencing technology, and biological functions of differentially expressed RNAs were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Validation of several differentially expressed RNAs was performed using quantitative real-time PCR (qRT-PCR). Protein-protein interaction (PPI) analysis and competitive endogenous RNA (ceRNA) prediction were used to identify interactions among hub genes and the interaction among lncRNAs, miRNAs, and mRNAs. The results showed that compared with the normal group, there were 267 mRNAs and 329 lncRNAs differentially expressed in ACL and 726 mRNAs and 609 lncRNAs in MCL in the injured group. Compared with MCL, 420 mRNAs and 470 lncRNAs were differentially expressed in ACL in the normal group; 162 mRNAs and 205 lncRNAs were differentially expressed in ACL in the injured group. Several important lncRNAs and genes were identified, namely, COL7A1, LIF, FGFR2, EPHA2, CSF1, MMP2, MMP9, SOX5, LOX, MSTRG.1737.1, MSTRG.26038.25, MSTRG.20209.5, MSTRG.22764.1, and MSTRG.18113.1, which are closely related to inflammatory response, tissue damage repair, cell proliferation, differentiation, migration, and apoptosis. Further study of the functions of these genes may help to better understand the specific molecular mechanisms underlying the occurrence of endogenous repair disorders in ACL, which may provide new ideas for further exploration of effective means to promote endogenous repair of ACL injury.

The endogenous progenitor response following traumatic brain injury: a target for cell therapy paradigms.

Although there is ample evidence that central nervous system progenitor pools respond to traumatic brain injury, the reported effects are variable and likely contribute to both recovery as well as pathophysiology. Through a better understanding of the diverse progenitor populations in the adult brain and their niche-specific reactions to traumatic insult, treatments can be tailored to enhance the benefits and dampen the deleterious effects of this response. This review provides an overview of endogenous precursors, the associated effects on cognitive recovery, and the potential of exogenous cell therapeutics to modulate these endogenous repair mechanisms. Beyond the hippocampal dentate gyrus and subventricular zone of the lateral ventricles, more recently identified sites of adult neurogenesis, the meninges, as well as circumventricular organs, are also discussed as targets for endogenous repair. Importantly, this review highlights that progenitor proliferation alone is no longer a meaningful outcome and studies must strive to better characterize precursor spatial localization, transcriptional profile, morphology, and functional synaptic integration. With improved insight and a more targeted approach, the stimulation of endogenous neurogenesis remains a promising strategy for recovery following traumatic brain injury.

High-Efficiency Multi-site Genomic Editing (HEMSE) Made Easy.

The ability to engineer bacterial genomes in an efficient way is crucial for many bio-related technologies. Single-stranded (ss) DNA recombineering technology allows to introduce mutations within bacterial genomes in a very simple and straightforward way. This technology was initially developed for E. coli but was later extended to other organisms of interest, including the environmentally and metabolically versatile Pseudomonas putida. The technology is based on three pillars: (1) adoption of a phage recombinase that works effectively in the target strain, (2) ease of introduction of short ssDNA oligonucleotide that carries the mutation into the bacterial cells at stake and (3) momentary suppression of the endogenous mismatch repair (MMR) through transient expression of a dominant negative mutL allele. In this way, the recombinase protects the ssDNA and stimulates recombination, while MutLE36KPP temporarily inhibits the endogenous MMR system, thereby allowing the introduction of virtually any possible type of genomic edits. In this chapter, a protocol is detailed for easily performing recombineering experiments aimed at entering single and multiple changes in the chromosome of P. putida. This was made by implementing the workflow named High-Efficiency Multi-site genomic Editing (HEMSE), which delivers simultaneous mutations with a simple and effective protocol.

Modulation of Inflamed Synovium Improves Migration of Mesenchymal Stromal Cells in Vitro Through Anti-Inflammatory Macrophages.

ObjectiveInflammation is known to negatively affect cartilage repair. However, it is unclear how inflammation influences the migration of mesenchymal stromal cells (MSCs) from the underlying bone marrow into the defect. We therefore aimed to investigate how synovial inflammation influences MSC migration, and whether modulation of inflammation with triamcinolone acetonide (TAA) may influence migration.DesignInflamed human osteoarthritic synovium, M(IFNγ+TNFα) pro-inflammatory macrophages, M(IL4) repair macrophages, M(IL10) anti-inflammatory macrophages, or synovial fibroblasts were cultured with/without TAA. Conditioned medium (CM) was harvested after 24 hours, and the effect on MSC migration was studied using a Boyden chamber assay. Inflammation was evaluated with gene expression and flow cytometry analysis.ResultsSynovium CM increased MSC migration. Modulation of synovial inflammation with TAA further increased migration 1.5-fold (P < 0.01). TAA significantly decreased TNFA, IL1B, and IL6 gene expression in synovium explants and increased CD163, a gene associated with anti-inflammatory macrophages. TAA treatment decreased the percentage of CD14+/CD80+ and CD14+/CD86+ pro-inflammatory macrophages and increased the percentage of CD14+/CD163+ anti-inflammatory macrophages in synovium explants. Interestingly, MSC migration was specifically enhanced by medium conditioned by M(IL4) macrophages and by M(IL10) macrophages treated with TAA, and unaffected by CM from M(IFNγ+TNFα) macrophages and synovial fibroblasts.ConclusionMacrophages secrete factors that stimulate the migration of MSCs. Modulation with TAA increased specifically the ability of anti-inflammatory macrophages to stimulate migration, indicating that they play an important role in secreting factors to attract MSCs. Modulating inflammation and thereby improving migration could be used in approaches based on endogenous repair of full-thickness cartilage defects.

Cas9-Mediated Targeted Mutagenesis in Plants.

Genome engineering technologies enable targeted mutations to be induced at almost any location in plant genomes. In particular, Cas9 nucleases use easily recoded RNA guides to target user-defined sequences and generate double-stranded breaks (DSB) that are then repaired by the cell's endogenous repair mechanisms. Incorrect repair results in mutations at the target. When the targets are in coding sequences, this often results in loss-of-function mutations. In this chapter, we describe a method to rapidly design and assemble RNA-guided Cas9 constructs for plants and test their ability to induce mutations at their intended targets in rapid assays using both Agrobacterium-mediated transient expression and PEG-mediated DNA delivery to protoplasts, the latter of which can be adapted to a wide range of plant species. We describe a PCR-based method for detecting mutagenesis and outline the steps required to segregate the Cas9 transgene from the targeted mutation to enable the production of transgene-free mutated plants. These techniques are amenable to a range of plant species and should accelerate the application of Cas-9-mediated genome engineering for basic plant science as well as crop development.

A role for insulin-like growth factor-1 in hippocampal plasticity following traumatic brain injury.

Traumatic brain injury (TBI) initiates a constellation of secondary injury cascades, leading to neuronal damage and dysfunction that is often beyond the scope of endogenous repair mechanisms. Cognitive deficits are among the most persistent morbidities resulting from TBI, necessitating a greater understanding of mechanisms of posttraumatic hippocampal damage and neuroplasticity and identification of therapies that improve recovery by enhancing repair pathways. Focusing here on hippocampal neuropathology associated with contusion-type TBIs, the impact of brain trauma on synaptic structure and function and the process of adult neurogenesis is discussed, reviewing initial patterns of damage as well as evidence for spontaneous recovery. A case is made that insulin-like growth factor-1 (IGF-1), a growth-promoting peptide synthesized in both the brain and the periphery, is well suited to augment neuroplasticity in the injured brain. Essential during brain development, multiple lines of evidence delineate roles in the adult brain for IGF-1 in the maintenance of synapses, regulation of neurotransmission, and modulation of forms of synaptic plasticity such as long-term potentiation. Further, IGF-1 enhances adult hippocampal neurogenesis though effects on proliferation and neuronal differentiation of neural progenitor cells and on dendritic growth of newly born neurons. Post-injury administration of IGF-1 has been effective in rodent models of TBI in improving learning and memory, attenuating death of mature hippocampal neurons and promoting neurogenesis, providing critical proof-of-concept data. More studies are needed to explore the effects of IGF-1-based therapies on synaptogenesis and synaptic plasticity following TBI and to optimize strategies in order to stimulate only appropriate, functional neuroplasticity.

Endogenous repair theory enriches construction strategies for orthopaedic biomaterials: a narrative review.

ABSTRACTThe development of tissue engineering has led to new strategies for mitigating clinical problems; however, the design of the tissue engineering materials remains a challenge. The limited sources and inadequate function, potential risk of microbial or pathogen contamination, and high cost of cell expansion impair the efficacy and limit the application of exogenous cells in tissue engineering. However, endogenous cells in native tissues have been reported to be capable of spontaneous repair of the damaged tissue. These cells exhibit remarkable plasticity, and thus can differentiate or be reprogrammed to alter their phenotype and function after stimulation. After a comprehensive review, we found that the plasticity of these cells plays a major role in establishing the cell source in the mechanism involved in tissue regeneration. Tissue engineering materials that focus on assisting and promoting the natural self-repair function of endogenous cells may break through the limitations of exogenous seed cells and further expand the applications of tissue engineering materials in tissue repair. This review discusses the effects of endogenous cells, especially stem cells, on injured tissue repairing, and highlights the potential utilisation of endogenous repair in orthopaedic biomaterial constructions for bone, cartilage, and intervertebral disc regeneration.

Stem cell-derived biofactors fight against coronavirus infection.

Despite various treatment protocols and newly recognized therapeutics, there are no effective treatment approaches against coronavirus disease. New therapeutic strategies including the use of stem cells-derived secretome as a cell-free therapy have been recommended for patients with critical illness. The pro-regenerative, pro-angiogenic, anti-inflammatory, anti-apoptotic, immunomodulatory, and trophic properties of stem cells-derived secretome, extracellular vesicles (EVs), and bioactive factors have made them suitable candidates for respiratory tract regeneration in coronavirus disease 2019 (COVID-19) patients. EVs including microvesicles and exosomes can be applied for communication at the intercellular level due to their abilities in the long-distance transfer of biological messages such as mRNAs, growth factors, transcription factors, microRNAs, and cytokines, and therefore, simulate the specifications of the parent cell, influencing target cells upon internalization and/or binding. EVs exhibit both anti-inflammatory and tolerogenic immune responses by regulation of proliferation, polarization, activation, and migration of different immune cells. Due to effective immunomodulatory and high safety including a minimum risk of immunogenicity and tumorigenicity, mesenchymal stem cell (MSC)-EVs are more preferable to MSC-based therapies. Thus, as an endogenous repair and inflammation-reducing agent, MSC-EVs could be used against COVID-19 induced morbidity and mortality after further mechanistic and preclinical/clinical investigations. This review is focused on the therapeutic perspective of the secretome of stem cells in alleviating the cytokine storm and organ injury in COVID-19 patients.

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.

Protocols for marker-free gene knock-out and knock-down in Kluyveromyces marxianus using CRISPR/Cas9.

ABSTRACTThere is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.

Citrullinated myelin induces microglial TNF\u03b1 and inhibits endogenous repair in the cuprizone model of demyelination

BackgroundMicroglia are the primary phagocytes of the central nervous system and are responsible for removing damaged myelin following demyelination. Previous investigations exploring the consequences of myelin phagocytosis on microglial activation overlooked the biochemical modifications present on myelin debris. Such modifications, including citrullination, are increased within the inflammatory environment of multiple sclerosis lesions.MethodsMouse cortical myelin isolated by ultracentrifugation was citrullinated ex vivo by incubation with the calcium-dependent peptidyl arginine deiminase PAD2. Demyelination was induced by 6 weeks of cuprizone (0.3%) treatment and spontaneous repair was initiated by reversion to normal chow. Citrullinated or unmodified myelin was injected into the primary motor cortex above the cingulum bundle at the time of reversion to normal chow and the consequent impact on remyelination was assessed by measuring the surface area of myelin basic protein-positive fibers in the cortex 3 weeks later. Microglial responses to myelin were characterized by measuring cytokine release, assessing flow cytometric markers of microglial activation, and RNAseq profiling of transcriptional changes.ResultsCitrullinated myelin induced a unique microglial response marked by increased tumor necrosis factor α (TNFα) production both in vitro and in vivo. This response was not induced by unmodified myelin. Injection of citrullinated myelin but not unmodified myelin into the cortex of cuprizone-demyelinated mice significantly inhibited spontaneous remyelination. Antibody-mediated neutralization of TNFα blocked this effect and restored remyelination to normal levels.ConclusionsThese findings highlight the role of post-translation modifications such as citrullination in the determination of microglial activation in response to myelin during demyelination. The inhibition of endogenous repair induced by citrullinated myelin and the reversal of this effect by neutralization of TNFα may have implications for therapeutic approaches to patients with inflammatory demyelinating disorders.

Mobilizing Endogenous Repair Through Understanding Immune Reaction With Biomaterials.

With few exceptions, humans are incapable of fully recovering from severe physical trauma. Due to these limitations, the field of regenerative medicine seeks to find clinically viable ways to repair permanently damaged tissue. There are two main approaches to regenerative medicine: promoting endogenous repair of the wound, or transplanting a material to replace the injured tissue. In recent years, these two methods have fused with the development of biomaterials that act as a scaffold and mobilize the body’s natural healing capabilities. This process involves not only promoting stem cell behavior, but by also inducing activity of the immune system. Through understanding the immune interactions with biomaterials, we can understand how the immune system participates in regeneration and wound healing. In this review, we will focus on biomaterials that promote endogenous tissue repair, with discussion on their interactions with the immune system.

Heart stem cells: hope or myth?

The search and study of endogenous heart repair remains an urgent issue in modern regenerative medicine. It is generally accepted that the human heart has a limited regenerative potential, but recent studies show that functionally significant regeneration is possible. However, the mechanisms underlying these processes remain poorly understood. In the heart, there are populations of resident mesenchymal cells that have some properties of stem cells that carry certain markers, such as c-kit+, Sca-1, etc. The ability of these cells to differentiate directly into cardiomyocytes remains controversial, but their use in clinical trials has shown improved cardiac function in patients with myocardial infarction. Currently, approaches are being developed to use, mainly, induced pluripotent stem cells as a promising regenerative therapy, but the cardioprotective role of cardiac mesenchymal cells remains the subject of active study due to their paracrine signaling.

Combined administration of laminin-221 and prostacyclin agonist enhances endogenous cardiac repair in an acute infarct rat heart

Although endogenous cardiac repair by recruitment of stem cells may serve as a therapeutic approach to healing a damaged heart, how to effectively enhance the migration of stem cells to the damaged heart is unclear. Here, we examined whether the combined administration of prostacyclin agonist (ONO1301), a multiple-cytokine inducer, and stem cell niche laminin-221 (LM221), enhances regeneration through endogenous cardiac repair. We administered ONO1301- and LM221-immersed sheets, LM221-immersed sheets, ONO1301-immersed sheets, and PBS-immersed sheets (control) to an acute infarction rat model. Four weeks later, cardiac function, histology, and cytokine expression were analysed. The combined administration of LM221 and ONO1301 upregulated angiogenic and chemotactic factors in the myocardium after 4 weeks and enhanced the accumulation of ILB4 positive cells, SMA positive cells, and platelet-derived growth factor receptor alpha (PDGFRα) and CD90 double-positive cells, leading to the generation of mature microvascular networks. Interstitial fibrosis reduced and functional recovery was prominent in LM221- and ONO1301-administrated hearts as compared with those in ONO1301-administrated or control hearts. LM221 and ONO1301 combination enhanced recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute myocardial infarction hearts, highlighting a new promising acellular approach for the failed heart.

Device-Based Enrichment of Knee Joint Synovial Cells to Drive MSC Chondrogenesis Without Prior Culture Expansion In Vitro: A Step Closer to 1-Stage Orthopaedic Procedures

Background: Synovial fluid (SF) mesenchymal stem cells (MSCs) are derived from the synovial membrane and have cartilage repair potential. Their current use in clinical practice is largely exploratory. As their numbers tend to be small, therapeutic procedures using MSCs typically require culture expansion. Previous reports indicate that the stem cell–mobilizing device (STEM device) intraoperatively increases SF-MSCs. Purpose: This study evaluated the chondrogenic potential of non–culture expanded synovium-mobilized MSCs and SF-microfragments obtained after enrichment using the STEM device and ascertained if device-mediated synovial membrane manipulation facilitated ongoing MSC release. Study Design: Controlled laboratory study. Methods: Two samples of aspiration fluid were collected intraoperatively before and after STEM device utilization from patients (n = 16) undergoing diagnostic or therapeutic knee arthroscopy. Human knee synovium (n = 5) was collected during total knee replacement, and a suspended culture was performed to assess the effect of the STEM device on ongoing MSC release. Colony forming unit–fibroblastic assays were used to determine the number of MSCs. Additionally, cytometric characterization of stromal and immune cells and chondrogenesis differentiation assay were performed without culture expansion. Filtered platelet concentrates were prepared using the HemaTrate system. Results: After STEM device use, a significant increase was evident in SF-MSCs (P = .03) and synovial fluid–resident synovial tissue microfragments (P = .03). In vitro–suspended synovium released significantly more MSCs following STEM device use than nonstimulated synovium (P = .01). The STEM device–released total cellular fraction produced greater in vitro chondrogenesis with significantly more glycosaminoglycans (GAGs; P < .0001) when compared with non–STEM device synovial fluid material. Nonexpanded SF-MSCs and SF-microfragments combined with autologous filtered platelet concentrate produced significantly more GAGs than the complete chondrogenic media (P < .0001). The STEM device–mobilized cells contained more M2 macrophage cells and fewer M1 cells. Conclusion: Non–culture expanded SF-MSCs and SF-microfragments had the potential to undergo chondrogenesis without culture expansion, which can be augmented using the STEM device with increased MSC release from manipulated synovium for several days. Although preliminary, these findings offer proof of concept toward manipulation of the knee joint environment to facilitate endogenous repair responses. Clinical Relevance: Although numbers were small, this study highlights 3 factors relevant to 1-stage joint repair using the STEM device: increased SF-MSCs and SF-microfragments and prolonged synovial release of MSCs. Joint repair strategies involving endogenous MSCs for cartilage repair without the need for culture expansion in a 1-stage procedure may be possible.

Programmable Building of Radially Gradient Nanofibrous Patches Enables Deployment, Bursting Bearing Capability, and Stem Cell Recruitment

AbstractFibrous patches capable of withstanding bursting force and recruiting endogenous stem cells are of great demand for wound treatment. A programmable strategy for development of radially gradient nanofibrous patches with rapid deployment property, robust bursting bearing capability, and excellent mesenchymal stem cell (MSC) recruitment capability, is demonstrated. Benefiting from the royal water lily‐like radially branched architecture, the gradient fibrous (GF) patches exhibit fast deployment in aqueous solution (2 s), high bursting strength of 4.6 N, as well as “center‐to‐periphery” gradient immobilization of stromal‐cell‐derived factor 1α (SDF1α). The SDF1α gradient patches direct MSC migration from the periphery to the center along the aligned nanofibers, resulting in a 4.2 times higher migrated cell number and 2.6 times greater maximum migration distance than random fibrous patches with homogenous SDF1α. The gelatin methacryloyl coated GF patches respond to matrix metalloproteinase‐9 for “on‐demand” release of anti‐inflammatory drug diclofenac sodium (DS). Furthermore, repair of the mouse full‐thickness skin incision validates that SDF1α/DS/GF patches are able to provide feasible microenvironment to attenuate inflammation and improve endogenous MSC recruitment, leading to accelerated wound healing. This work may open a new pathway for development of smart tough fibrous patches for stimulating endogenous repair mechanisms during tissue regeneration.