A New Bioactive Fibrin Formulation Provided Superior Cartilage Regeneration in a Caprine Model.

The effective and long-term treatment of cartilage defects is an unmet need among patients worldwide. In the past, several synthetic and natural biomaterials have been designed to support functional articular cartilage formation. However, they have mostly failed to enhance the terminal stage of chondrogenic differentiation, leading to scar tissue formation after the operation. Growth factors substantially regulate cartilage regeneration by acting on receptors to trigger intracellular signaling and cell recruitment for tissue regeneration. In this study, we investigated the effect of recombinant insulin-like growth factor 1 (rIGF-1), loaded in fibrin microbeads (FibIGF1), on cartilage regeneration. rIGF-1-loaded fibrin microbeads were injected into full-thickness cartilage defects in the knees of goats. The stability, integration, and quality of tissue repair were evaluated at 1 and 6 months by gross morphology, histology, and collagen type II staining. The in vivo results showed that compared to plain fibrin samples, particularly at 6 months, FibIGF1 improved the functional cartilage formation, confirmed through gross morphology, histology, and collagen type II immunostaining. FibIGF1 could be a promising candidate for cartilage repair in the clinic.

RANK-RANKL-OPG expression after gingival mesenchymal stem cell hypoxia preconditioned application in an orthodontic tooth movement animal model.

Background: The expression of receptor activator of Nuclear Factor Kappa Beta (RANK) and its ligand (RANKL), as well as osteoprotegrin (OPG), in the alveolar bone (AB), may improve bone remodeling during orthodontic tooth movement (OTM). It is hypothesized that hypoxia-preconditioned gingival mesenchymal stem cells (GMSC) may be more effective than normoxia-preconditioned GMSC in this regard. This study aims to investigate the expression of RANK, RANKL, and OPG in the compression and tension sides of AB after allogeneic administration of GMSC that were normoxia or hypoxia-preconditioned in rabbits (Oryctolagus cuniculus) undergoing OTM. Methods: Twenty-four healthy young male rabbits were divided into two groups: T1, which underwent OTM and received normoxia-preconditioned GMSC, and T2, which underwent OTM and received hypoxia-preconditioned GMSC. A ligature wire was attached to the mandibular first molar and connected to a 50 g/mm2 closed coil spring, exerting force on the central incisor and left mandibular molar of the experimental animals. After 24 h of OTM, either normoxia- or hypoxia-preconditioned GMSC were injected into the gingiva of the samples in a single dose of 20 µl of phosphate-buffered saline (PBS). All samples were sacrificed on days 7, 14, and 28, and immunohistochemistry was performed to analyze the expression of RANK, RANKL, and OPG on the tension and compression sides. Results: The expressions of RANK-RANKL-OPG in the alveolar bone of the compression and tension sides were significantly different during the 14-day period of OTM following allogeneic administration of GMSC that were normoxia or hypoxia-preconditioned (p < 0.05). Conclusion: The expression of RANK-RANKL was significantly increased on the compression side of the alveolar bone during OTM after the administration of hypoxia-preconditioned allogeneic GMSC but not on the tension side. Conversely, RANKL-OPG expression was enhanced on the tension side but not on the compression side, as observed through immunohistochemical analysis in vivo.

Gingiva Mesenchymal Stem Cells Normoxic or Hypoxic Preconditioned Application Under Orthodontic Mechanical Force on Osterix, Osteopontin, and ALP Expression.

OBJECTIVES: The aim of this article was to investigate Osterix, ALP, and osteopontin expression in the compression and tension sides of alveolar bone after the application of normoxic/hypoxic-preconditioned GMSCs in rabbits (Oryctolagus cuniculus) induced with OMF. MATERIALS AND METHODS: Forty-eight healthy, young male rabbits were divided into four groups: [-] OMF; [+] OMF; OMF with GMSCs normoxic-preconditioned; and OMF and GMSCs hypoxic-preconditioned. The central incisor and left mandibular molar in the experimental animals were moved, the mandibular first molar was moved mesially using nickel titanium (NiTi) and stainless steel ligature wire connected to a 50 g/mm2 light force closed coil spring. Allogeneic application of normoxic or hypoxic-preconditioned GMSCs was used in as many as 106 cells in a 20 µL phosphate buffered saline single dose and injected into experimental animals' gingiva after 1 day of OTM. On days 7, 14, and 28, all experimental animals were euthanized. Osterix, ALP, and osteopontin expressions were examined by immunohistochemistry. RESULTS: Osterix, ALP, and osteopontin expressions were significantly different after allogeneic application of hypoxic-preconditioned GMSCs than normoxic-preconditioned GMSCs in the tension and compression of the alveolar bone side during OMF (p < 0.05). CONCLUSION: Osterix, ALP, and osteopontin expressions were significantly more enhanced post-transplantation of GMSCs with hypoxic-preconditioning than after transplantation of normoxic-preconditioned GMSCs in rabbits (O. cuniculus) induced with OMF.

The role of autophagy in mitigating osteoarthritis progression via regulation of chondrocyte apoptosis: A review.

Osteoarthritis (OA) is the most prevalent chronic joint disease with an immense socioeconomic burden; however, no treatment has achieved complete success in effectively halting or reversing cartilage degradation, which is the central pathophysiological feature of OA. Chondrocytes loss or dysfunction is a significant contributing factor to the progressive cartilage deterioration as these sole resident cells have a crucial role to produce extracellular matrix proteins, thus maintaining cartilage structure and homeostasis. It has been previously suggested that death of chondrocytes occurring through apoptosis substantially contributes to cartilage degeneration. Although the occurrence of apoptosis in osteoarthritic cartilage and its correlation with cartilage degradation is evident, the causes of chondrocyte apoptosis leading to matrix loss are still not well-understood. Autophagy, an intracellular degradative mechanism that eliminates dysfunctional cytoplasmic components to aid cell survival in unfavourable conditions, is a potential therapeutic target to inhibit chondrocyte apoptosis and reduce OA severity. Despite accumulating evidence indicating significant cytoprotective effects of autophagy against chondrocyte apoptosis, the mechanistic link between autophagy and apoptosis in chondrocytes remains to be further explored. In this review, we summarize the relevant mechanistic events that perpetuate chondrocyte apoptosis and highlight the prominent role of autophagy in modulating these events to mitigate OA progression.

The potential mechanism of hypoxia-induced tenogenic differentiation of mesenchymal stem cell for tendon regeneration.

Tendon injuries account up to 50% of all musculoskeletal problems and remains a challenge to treat owing to the poor intrinsic reparative ability of tendon tissues. The natural course of tendon healing is very slow and often leads to fibrosis and disorganized tissues with inferior biomechanical properties. Mesenchymal stem cells (MSC) therapy is a promising alternative strategy to augment tendon repair due to its proliferative and multilineage differentiation potential. Hypoxic conditioning of MSC have been shown to enhance their tenogenic differentiation capacity. However, the mechanistic pathway by which this is achieved is yet to be fully defined. A key factor involved in this pathway is hypoxia-inducible factor-1-alpha (HIF-1α). This review aims to discuss the principal mechanism underlying the enhancement of MSC tenogenic differentiation by hypoxic conditioning, particularly the central role of HIF-1α in mediating activation of tenogenic pathways in the MSC. We focus on the interaction between HIF-1α with fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) in regulating MSC tenogenic differentiation pathways in hypoxic conditions. Strategies to promote stabilization of HIF-1α either through direct manipulation of oxygen tension or the use of hypoxia mimicking agents are therefore beneficial in increasing the efficacy of MSC therapy for tendon repair.

Adipose and Bone Marrow Derived-Mesenchymal Stromal Cells Express Similar Tenogenic Expression Levels when Subjected to Mechanical Uniaxial Stretching In Vitro.

The present study was conducted to determine whether adipose derived mesenchymal stromal cells (AD-MSCs) or bone marrow derived-MSCs (BM-MSCs) would provide superior tenogenic expressions when subjected to cyclical tensile loading. The results for this would indicate the best choice of MSCs source to be used for cell-based tendon repair strategies. Both AD-MSCs and BM-MSCs were obtained from ten adult donors (N = 10) and cultured in vitro. At passaged-2, cells from both groups were subjected to cyclical stretching at 1 Hz and 8% of strain. Cellular morphology, orientation, proliferation rate, protein, and gene expression levels were compared at 0, 24, and 48 hours of stretching. In both groups, mechanical stretching results in similar morphological changes, and the redirection of cell alignment is perpendicular to the direction of stretching. Loading at 8% strain did not significantly increase proliferation rates but caused an increase in total collagen expression and tenogenic gene expression levels. In both groups, these levels demonstrated no significant differences suggesting that in a similar loading environment, both cell types possess similar tenogenic potential. In conclusion, AD-MSCs and BM-MSCs both demonstrate similar tenogenic phenotypic and gene expression levels when subjected to cyclic tensile loading at 1 Hz and 8% strain, thus, suggesting that the use of either cell source may be suitable for tendon repair.

Discovering the Potential of Natural Antioxidants in Age-Related Macular Degeneration: A Review.

Age-related macular degeneration (AMD) is a multifactorial disease associated with anatomical changes in the inner retina. Despite tremendous advances in clinical care, there is currently no cure for AMD. This review aims to evaluate the published literature on the therapeutic roles of natural antioxidants in AMD. A literature search of PubMed, Web of Science and Google Scholar for peer-reviewed articles published between 1 January 2011 and 31 October 2021 was undertaken. A total of 82 preclinical and 18 clinical studies were eligible for inclusion in this review. We identified active compounds, carotenoids, extracts and polysaccharides, flavonoids, formulations, vitamins and whole foods with potential therapeutic roles in AMD. We evaluated the integral cellular signaling pathways including the activation of antioxidant pathways and angiogenesis pathways orchestrating their mode of action. In conclusion, we examined the therapeutic roles of natural antioxidants in AMD which warrant further study for application in clinical practice. Our current understanding is that natural antioxidants have the potential to improve or halt the progression of AMD, and tailoring therapeutics to the specific disease stages may be the key to preventing irreversible vision loss.

Mechanical Strain-Mediated Tenogenic Differentiation of Mesenchymal Stromal Cells Is Regulated through Epithelial Sodium Channels.

It has been suggested that mechanical strain may elicit cell differentiation in adult somatic cells through activation of epithelial sodium channels (ENaC). However, such phenomenon has not been previously demonstrated in mesenchymal stromal cells (MSCs). The present study was thus conducted to investigate the role of ENaC in human bone marrow-derived MSCs (hMSCs) tenogenic differentiation during uniaxial tensile loading. Passaged-2 hMSCs were seeded onto silicone chambers coated with collagen I and subjected to stretching at 1 Hz frequency and 8% strain for 6, 24, 48, and 72 hours. Analyses at these time points included cell morphology and alignment observation, immunocytochemistry and immunofluorescence staining (collagen I, collagen III, fibronectin, and N-cadherin), and gene expression (ENaC subunits, and tenogenic markers). Unstrained cells at similar time points served as the control group. To demonstrate the involvement of ENaC in the differentiation process, an ENaC blocker (benzamil) was used and the results were compared to the noninhibited hMSCs. ENaC subunits' (α, ß, γ, and δ) expression was observed in hMSCs, although only α subunit was significantly increased during stretching. An increase in tenogenic genes' (collagen1, collagen3, decorin, tenascin-c, scleraxis, and tenomodulin) and proteins' (collagen I, collagen III, fibronectin, and N-cadherin) expression suggests that hMSCs underwent tenogenic differentiation when subjected to uniaxial loading. Inhibition of ENaC function resulted in decreased expression of these markers, thereby suggesting that ENaC plays a vital role in tenogenic differentiation of hMSCs during mechanical loading.

Effect of Chitosan Nanoparticle-Loaded Thymus serpyllum on Hydrogen Peroxide-Induced Bone Marrow Stromal Cell Damage.

We have determined the protective effects of Thymus serpyllum (TS) extract and nanoparticle-loaded TS on hydrogen peroxide-induced cell death of mesenchymal stromal cells (MSCs) in vitro. Gas chromatography-mass spectroscopy confirmed the spectrum of active components in the extract. Out of the three different extracts, the hexane extract showed significant free radical scavenging activity. Treatment of MSCs with H2O2 (hydrogen peroxide) significantly increased intracellular cell death; however, pretreatment with TS extract and nanoparticle-loaded TS (200 µg/ml) suppressed H2O2-induced elevation of Cyt-c and MMP13 and increased the survival rates of MSCs. H2O2-induced (0.1 mM) changes in cytokines were attenuated in the extract and nanoparticles by pretreatment and cotreatment at two time points (p < 0.05). H2O2 increased cell apoptosis. In contrast, treatment with nanoparticle-loaded TS suppressed the percentage of apoptosis considerably (p < 0.05). Therefore, TS may be considered as a potential candidate for enhancing the effectiveness of MSC transplantation in cell therapy.

Electrospin-Coating of Paper: A Natural Extracellular Matrix Inspired Design of Scaffold.

Paper has recently found widespread applications in biomedical fields, especially as an alternative scaffolding material for cell cultures, owing to properties such as its fibrous nature, porosity and flexibility. However, paper on its own is not an optimal material for cell cultures as it lacks adhesion moieties specific to mammalian cells, and modifications such as hydrogel integration and chemical vapor deposition are necessary to make it a favorable scaffolding material. The present study focuses on modification of filter paper through electrospin-coating and dip-coating with polycaprolactone (PCL), a promising biomaterial in tissue engineering. Morphological analysis, evaluation of cell viability, alkaline phosphatase (ALP) activity and live/dead assays were conducted to study the potential of the modified paper-based scaffold. The results were compared to filter paper (FP) and electrospun PCL (ES-PCL) as reference samples. The results indicate that electrospin-coating paper is a simple and efficient way of modifying FP. It not only improves the morphology of the deposited electrospun layer through reduction of the fiber diameter by nearly 75%, but also greatly reduces the scaffold fabrication time compared to ES-PCL. The biochemical assays (Resazurin and ALP) indicate that electrospin-coated filter paper (ES-PCL/FP) provides significantly higher readings compared to all other groups. The live/dead results also show improved cell-distribution and cell-scaffold attachment all over the ES-PCL/FP.

Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.

Fate of tenogenic differentiation potential of human bone marrow stromal cells by uniaxial stretching affected by stretch-activated calcium channel agonist gadolinium.

The role for mechanical stimulation in the control of cell fate has been previously proposed, suggesting that there may be a role of mechanical conditioning in directing mesenchymal stromal cells (MSCs) towards specific lineage for tissue engineering applications. Although previous studies have reported that calcium signalling is involved in regulating many cellular processes in many cell types, its role in managing cellular responses to tensile loading (mechanotransduction) of MSCs has not been fully elucidated. In order to establish this, we disrupted calcium signalling by blocking stretch-activated calcium channel (SACC) in human MSCs (hMSCs) in vitro. Passaged-2 hMSCs were exposed to cyclic tensile loading (1 Hz + 8% for 6, 24, 48, and 72 hours) in the presence of the SACC blocker, gadolinium. Analyses include image observations of immunochemistry and immunofluorescence staining from extracellular matrix (ECM) production, and measuring related tenogenic and apoptosis gene marker expression. Uniaxial tensile loading increased the expression of tenogenic markers and ECM production. However, exposure to strain in the presence of 20 µM gadolinium reduced the induction of almost all tenogenic markers and ECM staining, suggesting that SACC acts as a mechanosensor in strain-induced hMSC tenogenic differentiation process. Although cell death was observed in prolonged stretching, it did not appear to be apoptosis mediated. In conclusion, the knowledge gained in this study by elucidating the role of calcium in MSC mechanotransduction processes, and that in prolonged stretching results in non-apoptosis mediated cell death may be potential useful for regenerative medicine applications.

The proliferation and tenogenic differentiation potential of bone marrow-derived mesenchymal stromal cell are influenced by specific uniaxial cyclic tensile loading conditions.

It has been previously demonstrated that mechanical stimuli are important for multipotent human bone marrow-derived mesenchymal stromal cells (hMSCs) to maintain good tissue homeostasis and even to enhance tissue repair processes. In tendons, this is achieved by promoting the cellular proliferation and tenogenic expression/differentiation. The present study was conducted to determine the optimal loading conditions needed to achieve the best proliferation rates and tenogenic differentiation potential. The effects of mechanical uniaxial stretching using different rates and strains were performed on hMSCs cultured in vitro. hMSCs were subjected to cyclical uniaxial stretching of 4, 8 or 12 % strain at 0.5 or 1 Hz for 6, 24, 48 or 72 h. Cell proliferation was analyzed using alamarBlue[Formula: see text] assay, while hMSCs differentiation was analyzed using total collagen assay and specific tenogenic gene expression markers (type I collagen, type III collagen, decorin, tenascin-C, scleraxis and tenomodulin). Our results demonstrate that the highest cell proliferation is observed when 4 % strain [Formula: see text] 1 Hz was applied. However, at 8 % strain [Formula: see text] 1 Hz loading, collagen production and the tenogenic gene expression were highest. Increasing strain or rates thereafter did not demonstrate any significant increase in both cell proliferation and tenogenic differentiation. In conclusion, our results suggest that 4 % [Formula: see text] 1 Hz cyclic uniaxial loading increases cell proliferation, but higher strains are required for superior tenogenic expressions. This study suggests that selected loading regimes will stimulate tenogenesis of hMSCs.

The effects of staged intra-articular injection of cultured autologous mesenchymal stromal cells on the repair of damaged cartilage: a pilot study in caprine model.

INTRODUCTION: Treatment of chondral injuries remains a major issue despite the many advances made in cartilage repair techniques. Although it has been postulated that the use of marrow stimulation in combination with cell-based therapy may provide superior outcome, this has yet to be demonstrated. A pilot study was thus conducted to determine if bone marrow derived mesenchymal stromal cells (BM-MSCs) have modulatory effects on the repair outcomes of bone marrow stimulation (BMS) techniques. METHODS: Two full-thickness chondral 5 mm diameter defects were created in tandem on the medial condyle of left stifle joints of 18 Boer caprine (N = 18). Goats were then divided equally into three groups. Simultaneously, bone marrow aspirates were taken from the iliac crests from the goats in Group 1 and were sent for BM-MSC isolation and expansion in vitro. Six weeks later, BMS surgery, which involves subchondral drilling at the defect sites, was performed. After two weeks, the knees in Group 1 were given autologous intra-articular BM-MSCs (N = 6). In Group 2, although BMS was performed there were no supplementations provided. In Group 3, no intervention was administered. The caprines were sacrificed after six months. Repairs were evaluated using macroscopic assessment through the International Cartilage Repair Society (ICRS) scoring, histologic grading by O'Driscoll score, biochemical assays for glycosaminoglycans (GAGs) and gene expressions for aggrecan, collagen II and Sox9. RESULTS: Histological and immunohistochemical analyses demonstrated hyaline-like cartilage regeneration in the transplanted sites particularly in Group 1. In contrast, tissues in Groups 2 and 3 demonstrated mainly fibrocartilage. The highest ICRS and O'Driscoll scorings was also observed in Group 1, while the lowest score was seen in Group 3. Similarly, the total GAG/total protein as well as chondrogenic gene levels were expressed in the same order, that is highest in Group 1 while the lowest in Group three. Significant differences between these 3 groups were observed (P <0.05). CONCLUSIONS: This study suggests that supplementing intra-articular injections of BM-MSCs following BMS knee surgery provides superior cartilage repair outcomes.

First series of Exeter small stem primary total hip arthroplasty minimum 5 years of follow-up.

We carried out a prospective study of 47 Exeter (Stryker Inc, Warsaw, Ind) small stem total hip arthroplasty in 42 patients with an average age of 58 years and a mean follow-up of 8.5 years. The Oxford hip score improved from a preoperative mean of 47 to 17 at last follow-up. More than 87% patients had excellent or good Harris hip scores, and 90% were able to walk with little or no pain. Stem subsidence within the cement mantle was observed in 26% of cases, and none showed evidence of aseptic loosening or implant failure. Two stems were removed due to infection. The survival rate of this implant was 95.7% at 10 years. This first series of Exeter small stem showed excellent medium-term results, comparable to its larger counterparts.