Fibroblast growth factor 8b (FGF-8b) enhances myogenesis and inhibits adipogenesis in rotator cuff muscle cell populations in vitro.

Fatty expansion is one of the features of muscle degeneration due to muscle injuries, and its presence interferes with muscle regeneration. Specifically, poor clinical outcomes have been linked to fatty expansion in rotator cuff tears and repairs. Our group recently found that fibroblast growth factor 8b (FGF-8b) inhibits adipogenic differentiation and promotes myofiber formation of mesenchymal stem cells in vitro. This led us to hypothesize that FGF-8b could similarly control the fate of muscle-specific cell populations derived from rotator cuff muscle involved in muscle repair following rotator cuff injury. In this study, we isolate fibro-adipogenic progenitor cells (FAPs) and satellite stem cells (SCs) from rat rotator cuff muscle tissue and analyzed the effects of FGF-8b supplementation. Utilizing a cell plating protocol, we successfully isolate FAPs-rich fibroblasts (FIBs) and SCs-rich muscle progenitor cells (MPCs). Subsequently, we demonstrate that FIB adipogenic differentiation can be inhibited by FGF-8b, while MPC myogenic differentiation can be enhanced by FGF-8b. We further demonstrate that phosphorylated ERK due to FGF-8b leads to the inhibition of adipogenesis in FIBs and SCs maintenance and myofiber formation in MPCs. Together, these findings demonstrate the powerful potential of FGF-8b for rotator cuff repair by altering the fate of muscle undergoing degeneration.

Development of porcine skeletal muscle extracellular matrix-derived hydrogels with improved properties and low immunogenicity.

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

Muscle degeneration in chronic massive rotator cuff tears of the shoulder: Addressing the real problem using a graphene matrix.

Massive rotator cuff tears (MRCTs) of the shoulder cause disability and pain among the adult population. In chronic injuries, the tendon retraction and subsequently the loss of mechanical load lead to muscle atrophy, fat accumulation, and fibrosis formation over time. The intrinsic repair mechanism of muscle and the successful repair of the torn tendon cannot reverse the muscle degeneration following MRCTs. To address these limitations, we developed an electroconductive matrix by incorporating graphene nanoplatelets (GnPs) into aligned poly(l-lactic acid) (PLLA) nanofibers. This study aimed to understand 1) the effects of GnP matrices on muscle regeneration and inhibition of fat formation in vitro and 2) the ability of GnP matrices to reverse muscle degenerative changes in vivo following an MRCT. The GnP matrix significantly increased myotube formation, which can be attributed to enhanced intracellular calcium ions in myoblasts. Moreover, the GnP matrix suppressed adipogenesis in adipose-derived stem cells. These results supported the clinical effects of the GnP matrix on reducing fat accumulation and muscle atrophy. The histological evaluation showed the potential of the GnP matrix to reverse muscle atrophy, fat accumulation, and fibrosis in both supraspinatus and infraspinatus muscles at 24 and 32 wk after the chronic MRCTs of the rat shoulder. The pathological evaluation of internal organs confirmed the long-term biocompatibility of the GnP matrix. We found that reversing muscle degenerative changes improved the morphology and tensile properties of the tendon compared with current surgical techniques. The long-term biocompatibility and the ability of the GnP matrix to treat muscle degeneration are promising for the realization of MRCT healing and regeneration.

Injectable amnion hydrogel-mediated delivery of adipose-derived stem cells for osteoarthritis treatment.

Current treatment strategies for osteoarthritis (OA) predominantly address symptoms with limited disease-modifying potential. There is a growing interest in the use of adipose-derived stem cells (ADSCs) for OA treatment and developing biomimetic injectable hydrogels as cell delivery systems. Biomimetic injectable hydrogels can simulate the native tissue microenvironment by providing appropriate biological and chemical cues for tissue regeneration. A biomimetic injectable hydrogel using amnion membrane (AM) was developed which can self-assemble in situ and retain the stem cells at the target site. In the present study, we evaluated the efficacy of intraarticular injections of AM hydrogels with and without ADSCs in reducing inflammation and cartilage degeneration in a collagenase-induced OA rat model. A week after the induction of OA, rats were treated with control (phosphate-buffered saline), ADSCs, AM gel, and AM-ADSCs. Inflammation and cartilage regeneration was evaluated by joint swelling, analysis of serum by cytokine profiling and Raman spectroscopy, gross appearance, and histology. Both AM and ADSC possess antiinflammatory and chondroprotective properties to target the sites of inflammation in an osteoarthritic joint, thereby reducing the inflammation-mediated damage to the articular cartilage. The present study demonstrated the potential of AM hydrogel to foster cartilage tissue regeneration, a comparable regenerative effect of AM hydrogel and ADSCs, and the synergistic antiinflammatory and chondroprotective effects of AM and ADSC to regenerate cartilage tissue in a rat OA model.

Mechanically superior matrices promote osteointegration and regeneration of anterior cruciate ligament tissue in rabbits.

The gold standard treatment for anterior cruciate ligament (ACL) reconstruction is the use of tendon autografts and allografts. Limiting factors for this treatment include donor site morbidity, potential disease transmission, and variable graft quality. To address these limitations, we previously developed an off-the-shelf alternative, a poly(l-lactic) acid (PLLA) bioengineered ACL matrix, and demonstrated its feasibility to regenerate ACL tissue. This study aims to 1) accelerate the rate of regeneration using the bioengineered ACL matrix by supplementation with bone marrow aspirate concentrate (BMAC) and growth factors (BMP-2, FGF-2, and FGF-8) and 2) increase matrix strength retention. Histological evaluation showed robust tissue regeneration in all groups. The presence of cuboidal cells reminiscent of ACL fibroblasts and chondrocytes surrounded by an extracellular matrix rich in anionic macromolecules was up-regulated in the BMAC group. This was not observed in previous studies and is indicative of enhanced regeneration. Additionally, intraarticular treatment with FGF-2 and FGF-8 was found to suppress joint inflammation. To increase matrix strength retention, we incorporated nondegradable fibers, polyethylene terephthalate (PET), into the PLLA bioengineered ACL matrix to fabricate a "tiger graft." The tiger graft demonstrated the greatest peak loads among the experimental groups and the highest to date in a rabbit model. Moreover, the tiger graft showed superior osteointegration, making it an ideal bioengineered ACL matrix. The results of this study illustrate the beneficial effect bioactive factors and PET incorporation have on ACL regeneration and signal a promising step toward the clinical translation of a functional bioengineered ACL matrix.

Fish Models for Exploring Mitochondrial Dysfunction Affecting Neurodegenerative Disorders.

Neurodegenerative disorders are characterized by the progressive loss of neuronal structure or function, resulting in memory loss and movement disorders. Although the detailed pathogenic mechanism has not been elucidated, it is thought to be related to the loss of mitochondrial function in the process of aging. Animal models that mimic the pathology of a disease are essential for understanding human diseases. In recent years, small fish have become ideal vertebrate models for human disease due to their high genetic and histological homology to humans, ease of in vivo imaging, and ease of genetic manipulation. In this review, we first outline the impact of mitochondrial dysfunction on the progression of neurodegenerative diseases. Then, we highlight the advantages of small fish as model organisms, and present examples of previous studies regarding mitochondria-related neuronal disorders. Lastly, we discuss the applicability of the turquoise killifish, a unique model for aging research, as a model for neurodegenerative diseases. Small fish models are expected to advance our understanding of the mitochondrial function in vivo, the pathogenesis of neurodegenerative diseases, and be important tools for developing therapies to treat diseases.

Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates.

DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, we generated epigenomic profiles that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish and compared the profiles with those of human ES cells. We found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. We propose that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.

Targeted Ablation of Pancreatic ß Cells in Medaka.

The pancreas plays important roles in the regulation of blood glucose, and is a well-studied organ in mammals because its dysfunction causes serious disorders, such as diabetes mellitus. However, mammals have the limited capacity for tissue regeneration in their organs, including pancreas. Fish may be an attractive model for regeneration studies, as fish exhibit a greater capacity for regeneration than do mammals. To elucidate the regenerative capacity of pancreatic ß cells in medaka, we generated transgenic lines, in which ß cells can be specifically ablated using the nitroreductase (NTR)/metronidazole (Mtz) system. We examined ß-cell regeneration at embryonic-larval stages after specific ablation of ß cells, and found that medaka rapidly regenerate ß cells. Furthermore, we found that teleost-specific secondary islet have a unique feature in that their size increases in response to ß-cell ablation in principal islets.

Development of the pancreas in medaka, Oryzias latipes, from embryo to adult.

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1- and ptf1a-transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior-posterior axis.

Electroconductivity, a regenerative engineering approach to reverse rotator cuff muscle degeneration.

Muscle degeneration is one the main factors that lead to the high rate of retear after a successful repair of rotator cuff (RC) tears. The current surgical practices have failed to treat patients with chronic massive rotator cuff tears (RCTs). Therefore, regenerative engineering approaches are being studied to address the challenges. Recent studies showed the promising outcomes of electroactive materials (EAMs) on the regeneration of electrically excitable tissues such as skeletal muscle. Here, we review the most important biological mechanism of RC muscle degeneration. Further, the review covers the recent studies on EAMs for muscle regeneration including RC muscle. Finally, we will discuss the future direction toward the application of EAMs for the augmentation of RCTs.

Efficacy of a Novel Electroconductive Matrix To Treat Muscle Atrophy and Fat Accumulation in Chronic Massive Rotator Cuff Tears of the Shoulder.

The high retear rate after a successful repair of the rotator cuff (RC) is a major clinical challenge. Muscle atrophy and fat accumulation of RC muscles over time adversely affect the rate of retear. Since current surgical techniques do not improve muscle degenerative conditions, new treatments are being developed to reduce muscle atrophy and fat accumulation. In the previous study, we have shown the efficacy of aligned electroconductive nanofibrous fabricated by coating poly(3,4-ethylene dioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) nanoparticles onto aligned poly(ε-caprolactone) (PCL) electrospun nanofibers (PEDOT:PSS matrix) to reduce muscle atrophy in acute and subacute models of RC tears (RCTs). In this study, we further evaluated the efficacy of the PEDOT:PSS matrix to reduce muscle atrophy and fat accumulation in a rat model of chronic massive full-thickness RCTs (MRCTs). The matrices were transplanted on the myotendinous junction to the belly of the supraspinatus and infraspinatus muscles at 16 weeks after MRCTs. The biomechanics and histological assessments showed the potential of the PEDOT:PSS matrix to suppress the progression of muscle atrophy, fat accumulation, and fibrosis in both supraspinatus and infraspinatus muscles at 24 and 32 weeks after MRCTs. We also demonstrated that the PEDOT:PSS matrix implantation significantly improved the tendon morphology and tensile properties compared with current surgical techniques.

Overexpression of NDST1 Attenuates Fibrotic Response in Murine Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) hold tremendous potential for treating diseases and repairing damaged tissues. Heparan sulfate (HS) plays various roles in cellular signaling mechanisms. The importance of HS in stem cell function has been reported and well documented. However, there has been little progress in using HS for therapeutic purposes. We focused on one of the sulfotransferases, NDST1, which influences overall HS chain extent and sulfation pattern, with the expectation to enhance stem cell function by increasing the N-sulfation level. We herein performed transfections of a green fluorescent protein-vector control and NDST1-vector into mouse ADSCs to evaluate stem cell functions. Overexpression of NDST1 suppressed the osteogenic differentiation of ADSCs. There was no pronounced effect observed on the stemness, inflammatory gene expression, nor any noticeable effect in adipogenic and chondrogenic differentiation. Under the tumor necrosis factor-alpha stimulation, NDST1 overexpression induced several chemokine productions that attract neutrophils and macrophages. Finally, we identified an antifibrotic response in ADSCs overexpressing NDST1. This study provides a foundation for the evaluation of HS-related effects in ADSCs undergoing ex vivo gene manipulation.

Control of mesenchymal cell fate via application of FGF-8b in vitro.

In order to develop strategies to regenerate complex tissues in mammals, understanding the role of signaling in regeneration competent species and mammalian development is of critical importance. Fibroblast growth factor 8 (FGF-8) signaling has an essential role in limb morphogenesis and blastema outgrowth. Therefore, we aimed to study the effect of FGF-8b on the proliferation and differentiation of mesenchymal stem cells (MSCs), which have tremendous potential for therapeutic use of cell-based therapy. Rat adipose derived stem cells (ADSCs) and muscle progenitor cells (MPCs) were isolated and cultured in growth medium and various types of differentiation medium (osteogenic, chondrogenic, adipogenic, tenogenic, and myogenic medium) with or without FGF-8b supplementation. We found that FGF-8b induced robust proliferation regardless of culture medium. Genes related to limb development were upregulated in ADSCs by FGF-8b supplementation. Moreover, FGF-8b enhanced chondrogenic differentiation and suppressed adipogenic and tenogenic differentiation in ADSCs. Osteogenic differentiation was not affected by FGF-8b supplementation. FGF-8b was found to enhance myofiber formation in rat MPCs. Overall, this study provides foundational knowledge on the effect of FGF-8b in the proliferation and fate determination of MSCs and provides insight in its potential efficacy for musculoskeletal therapies.

Preparation and characterization of amnion hydrogel and its synergistic effect with adipose derived stem cells towards IL1ß activated chondrocytes.

Inflammation leads to chondrocyte senescence and cartilage degeneration, resulting in osteoarthritis (OA). Adipose-derived stem cells (ADSCs) exert paracrine effects protecting chondrocytes from degenerative changes. However, the lack of optimum delivery systems for ADSCs limits its use in the clinic. The use of extracellular matrix based injectable hydrogels has gained increased attention due to their unique properties. In the present study, we developed hydrogels from amnion tissue as a delivery system for ADSCs. We investigated the potential of amnion hydrogel to maintain ADSC functions, the synergistic effect of AM with ADSC in preventing the catabolic responses of inflammation in stimulated chondrocytes. We also investigated the role of Wnt/ß-catenin signaling pathway in IL-1ß induced inflammation in chondrocytes and the ability of AM-ADSC to inhibit Wnt/ß-catenin signaling. Our results showed that AM hydrogels supported cell viability, proliferation, and stemness. ADSCs, AM hydrogels and AM-ADSCs inhibited the catabolic responses of IL-1ß and inhibited the Wnt/ß-catenin signaling pathway, indicating possible involvement of Wnt/ß-catenin signaling pathways in IL-1ß induced inflammation. The results also showed that the synergistic effect of AM-ADSCs was more pronounced in preventing catabolic responses in activated chondrocytes. In conclusion, we showed that AM hydrogels can be used as a potential carrier for ADSCs, and can be developed as a potential therapeutic agent for treating OA.

Evaluation of a bioengineered ACL matrix's osteointegration with BMP-2 supplementation.

A poly (l-lactic) acid bioengineered anterior cruciate ligament (ACL) matrix has previously demonstrated the ability to support tissue regeneration in a rabbit ACL reconstruction model. The matrix was designed for optimal bone and ligament regeneration by developing a matrix with differential pore sizes in its bone and ligament compartments. Building upon past success, we designed a new bioengineered ACL matrix that is easier to install and can be used with endobutton fixation during ACL reconstruction. To achieve this, a new braiding procedure was developed to allow the matrix to be folded in half, making two-limbs, while maintaining its bone and ligament compartments. The osteointegration of the matrix with and without bone morphogenetic protein 2 (BMP-2) supplementation was evaluated in a rabbit ACL reconstruction model. Two doses of BMP-2 were evaluated, 1 and 10 µg, and delivered by saline injection into the bone tunnel at the end of surgery. A fibrous matrix-to-bone interface with occasional Sharpey's fibers was the primary mode of osteointegration observed. The matrix was also found to support a fibrocartilage matrix-to-bone interface. In some cases, the presence of chondrocyte-like cells was observed at the aperture of the bone tunnel and the center of the matrix within the bone tunnel. Treatment with BMP-2 was associated with a trend towards smaller bone tunnel cross-sectional areas, and 1 µg of BMP-2 was found to significantly enhance osteoid seam width in comparison with no BMP-2 or 10 µg of BMP-2 treatment. Regenerated tissue was well organized within the bioengineered ACL matrix and aligned with the poly (l-lactic) acid fibers. Disorganized tissue was found between the two-limbs of the bioengineered ACL matrix and hypothesized to be due to a lack of structural scaffolding. This study suggests that the bioengineered ACL matrix can undergo similar modes of osteointegration as current autografts and allografts, and that BMP-2 treatment may enhance osteoblastic activity within the bone tunnels.

The Axolotl Limb Regeneration Model as a Discovery Tool for Engineering the Stem Cell Niche.

PURPOSE OF REVIEW: Recent advances in genomics and gene editing have expanded the range of model organisms to include those with interesting biological capabilities such as regeneration. Among these are the classic models of regeneration biology, the salamander. Although stimulating endogenous regeneration in humans likely is many years away, with advances in stem cell biology and biomedical engineering (e.g. bio-inspired materials), it is evident that there is great potential to enhance regenerative outcomes by approaching the problem from an engineering perspective. The question at this point is what do we need to engineer? RECENT FINDINGS: The value of regeneration models is that they show us how regeneration works, which then can guide efforts to mimic these developmental processes therapeutically. Among these models, the Accessory Limb Model (ALM) was developed in the axolotl as a gain-of-function assay for the sequential steps that are required for successful regeneration. To date, this model has identified a number of proregenerative signals, including growth factor signaling associated with nerves, and signals associated with the extracellular matrix (ECM) that induce pattern formation. SUMMARY: Identification of these signals through the use of models in highly regenerative vertebrates (e.g. the axolotl) offers a wide range of possible modifications for engineering bio-inspired, biomimetic materials to create a dynamic stem cell niche for regeneration and scar-free repair.