Hyaluronidase inhibits TGF-ß-mediated rat periodontal ligament fibroblast expression of collagen and myofibroblast markers: An in vitro exploration of periodontal tissue remodeling.

OBJECTIVE: To determine the effect of hyaluronic acid (HA) degradation by hyaluronidase (HYAL) in inhibiting collagen fiber production by rat periodontal ligament cells (rPDLCs). DESIGN: Primary rPDLCs were isolated from the euthanized rats and used for in vitro experiments. The appropriate HYAL concentration was determined through CCK-8 testing for cytotoxicity detection and Alizarin red staining for mineralization detection. RT-qPCR and western blot assays were conducted to assess the effect of HYAL, with or without TGF-ß, on generation of collagen fiber constituents and expression of actin alpha 2, smooth muscle (ACTA2) of rPDLCs. RESULTS: Neither cell proliferation nor mineralization were significantly affected by treatment with 4 U/mL HYAL. HYAL (4 U/mL) alone downregulated type I collagen fiber (Col1a1 and Col1a2) and Acta2 mRNA expression; however, ACTA2 and COL1 protein levels were only downregulated by HYAL treatment after TGF-ß induction. CONCLUSIONS: Treatment of rPDLCs with HYAL can inhibit TGF-ß-induced collagen matrix formation and myofibroblast transformation.

Molecular histopathology of matrix proteins through autofluorescence super-resolution microscopy.

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training.

Recombinant human fibroblast growth factor 7 obtained from stable Chinese hamster ovary cells enhances wound healing.

Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control. Wound healing was most effective in the CP+FGF7 group. Particularly, on day 7 post-exposure, the CP+FGF7 and FGF7-only groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor. Epidermalization in H&E staining showed the same order of healing as hydroxyproline content. Additionally, the CP+FGF7 and FGF7-only group exhibited more notable blood vessel formation on days 7 and 14. In conclusion, the prepared FGF7 was effective in promoting wound healing and CHO cells can be a reliable platform for the mass production of FGF7.

Effect of Helichrysum italicum in Promoting Collagen Deposition and Skin Regeneration in a New Dynamic Model of Skin Wound Healing.

Natural products have many healing effects on the skin with minimal or no adverse effects. In this study, we analyzed the regenerative properties of a waste product (hydrolate) derived from Helichrysum italicum (HH) on scratch-tested skin cell populations seeded on a fluidic culture system. Helichrysum italicum has always been recognized in the traditional medicine of Mediterranean countries for its wide pharmacological activities. We recreated skin physiology with a bioreactor that mimics skin stem cell (SSCs) and fibroblast (HFF1) communication as in vivo skin layers. Dynamic culture models represent an essential instrument for recreating and preserving the complex multicellular organization and interactions of the cellular microenvironment. Both cell types were exposed to two different concentrations of HH after the scratch assay and were compared to untreated control cells. Collagen is the constituent of many wound care products that act directly on the damaged wound environment. We analyzed the role played by HH in stimulating collagen production during tissue repair, both in static and dynamic culture conditions, by a confocal microscopic analysis. In addition, we performed a gene expression analysis that revealed the activation of a molecular program of stemness in treated skin stem cells. Altogether, our results indicate a future translational application of this natural extract to support skin regeneration and define a new protocol to recreate a dynamic process of healing.

Investigating the Relevance of Cyclic Adenosine Monophosphate Response Element-Binding Protein to the Wound Healing Process: An In Vivo Study Using Photobiomodulation Treatment.

Monitoring inflammatory cytokines is crucial for assessing healing process and photobiomodulation (PBM) enhances wound healing. Meanwhile, cAMP response element-binding protein (CREB) is a regulator of cellular metabolism and proliferation. This study explored potential links between inflammatory cytokines and the activity of CREB in PBM-treated wounds. A total of 48 seven-week-old male SD rats were divided into four groups (wound location, skin or oral; treatment method, natural healing or PBM treatment). Wounds with a 6 mm diameter round shape were treated five times with an 808 nm laser every other day (total 60 J). The wound area was measured with a caliper and calculated using the elliptical formula. Histological analysis assessed the epidermal regeneration and collagen expression of skin and oral tissue with H&E and Masson's trichrome staining. Pro-inflammatory (TNF-α) and anti-inflammatory (TGF-ß) cytokines were quantified by RT-PCR. The ratio of phosphorylated CREB (p-CREB) to unphosphorylated CREB was identified through Western blot. PBM treatment significantly reduced the size of the wounds on day 3 and day 7, particularly in the skin wound group (p < 0.05 on day 3, p < 0.001 on day 7). The density of collagen expression was significantly higher in the PBM treatment group (in skin wound, p < 0.05 on day 3, p < 0.001 on day 7, and p < 0.05 on day 14; in oral wound, p < 0.01 on day 7). The TGF-ß/TNF-α ratio and the p-CREB/CREB ratio showed a parallel trend during wound healing. Our findings suggested that the CREB has potential as a meaningful marker to track the wound healing process.

Investigation of collagen reconstruction mechanism in skin wound through dual-beam laser welding: Insights from multi-spectroscopy, molecular dynamics simulation, and finite element multiphysics simulation.

Since the mechanism underlying real-time acquisition of mechanical strength during laser-induced skin wound fusion remains unclear, and collagen is the primary constituent of skin tissue, this study investigates the structural and mechanical alterations in collagen at temperatures ranging from 40 °C to 60 °C using various spectroscopic techniques and molecular dynamics calculations. The COMSOL Multiphysics coupling is employed to simulate the three-dimensional temperature field, stress-strain relationship, and light intensity distribution in the laser thermal affected zone of skin wounds during dual-beam laser welding process. Raman spectroscopy, synchronous fluorescence spectroscopy and circular dichroism measurement results confirm that laser energy activates biological activity in residues, leading to a transformation in the originally fractured structure of collagen protein for enhanced mechanical strength. Molecular dynamics simulations reveal that stable hydrogen bonds form at amino acid residues within the central region of collagen protein when the overall temperature peak around the wound reaches 60 °C, thereby providing stability to previously fractured skin incisions and imparting instantaneous strength. However, under a 55 °C system, Type I collagen ensures macrostructural stability while activating biological properties at amino acid bases to promote wound healing function; this finding aligns with experimental analysis results. The COMSOL simulation outcomes also correspond well with macroscopic morphology after laser welding samples, confirming that by maintaining temperatures between 55 °C-60 °C during laser welding of skin incisions not only can certain instantaneous mechanical strength be achieved but irreversible thermal damage can also be effectively controlled. It is anticipated that these findings will provide valuable insights into understanding the healing mechanism for laser-welded skin wounds.

The influence of immediate occlusal loading on micro/nano-structure of peri-implant jaw bone in rats.

PURPOSE: The objective of the present study was to ascertain the effect of immediate occlusal loading after implant placement on osseointegration and the micro/nanostructure of the surrounding bone. METHODS: After extraction of a rat maxillary right second molar, an implant was placed immediately with initial fixation (2 N< ). The implants were placed to avoid occlusal loading due to mastication, and in the loaded group, a superstructure was fabricated and subjected to occlusal loading. Bone morphometry, collagen fiber anisotropy, and biological apatite (BAp) crystallite alignment were quantitatively evaluated in both groups after extraction and fixation of the jaw bone at Days 7 and 21 after surgery. RESULTS: Osseointegration was observed in both groups. Bone morphometry showed significant differences in bone volume, trabecular number, trabecular thickness and bone mineral density (BMD) at Days 21 postoperatively (P < 0.05). A significant difference was also found in the trabecular separation at Days 7 postoperatively (P < 0.05). In the evaluation of collagen fiber anisotropy, collagen fiber bundles running differently from the existing bone were observed in both groups. In terms of BAp crystallite alignment, a specific structure was observed in the reconstructed new bone after implantation, and preferential orientation of BAp crystallite alignment was observed in the longitudinal direction of the implants in the Day 21 postoperative loaded group. CONCLUSION: When sufficient initial fixation is achieved at the time of dental implant placement, then the applied masticatory load may contribute to rapidly achieving not only bone volume, but also adequate bone quality after implant placement.

Compositional, Structural, and Biomechanical Properties of Three Different Soft Tissue-Hard Tissue Insertions: A Comparative Review.

Connective tissue attaches to bone across an insertion with spatial gradients in components, microstructure, and biomechanics. Due to regional stress concentrations between two mechanically dissimilar materials, the insertion is vulnerable to mechanical damage during joint movements and difficult to repair completely, which remains a significant clinical challenge. Despite interface stress concentrations, the native insertion physiologically functions as the effective load-transfer device between soft tissue and bone. This review summarizes tendon, ligament, and meniscus insertions cross-sectionally, which is novel in this field. Herein, the similarities and differences between the three kinds of insertions in terms of components, microstructure, and biomechanics are compared in great detail. This review begins with describing the basic components existing in the four zones (original soft tissue, uncalcified fibrocartilage, calcified fibrocartilage, and bone) of each kind of insertion, respectively. It then discusses the microstructure constructed from collagen, glycosaminoglycans (GAGs), minerals and others, which provides key support for the biomechanical properties and affects its physiological functions. Finally, the review continues by describing variations in mechanical properties at the millimeter, micrometer, and nanometer scale, which minimize stress concentrations and control stretch at the insertion. In summary, investigating the contrasts between the three has enlightening significance for future directions of repair strategies of insertion diseases and for bioinspired approaches to effective soft-hard interfaces and other tough and robust materials in medicine and engineering.

Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non-tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-ß1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-ß1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-ß1 signaling axis.

Does collagen cross linking have any effect on retinal circulation in patients with keratoconus? An optical coherence tomography angiography (OCTA) study.

BACKGROUND: We aimed to employ Optical Coherence Tomography Angiography (OCTA) to comprehensively assess changes in the optic nerve head (ONH) and macular perfusion before and after the Corneal Collagen Cross-Linking (CCL) procedure in patients with keratoconus. METHODS: A total of 22 keratoconus patient's candidate for CCL procedures were included based on specific criteria, with meticulous exclusion criteria in place to minimize potential confounders. Participants underwent OCTA assessments of the ONH and macula using the Spectralis OCT (Heidelberg) before CCL, as well as at 1- and 3-months post-CCL. MATLAB software was utilized for image analysis. RESULTS: The mean age of the participants was 20.09 ± 6.11, including 59% male, and the mean intraocular pressure (IOP) before the surgery was 13.59 ± 2.85 mmHg. Peripapillary Retinal nerve fiber layer (ppRNFL) thickness and overall retinal thickness remained stable post-CCL. However, significant alterations were observed in macular vessel density, emphasizing regional variations in vascular response. For macular large vessel density (LVD), both superficial and deep vascular complex (SVC and DVC) demonstrated significant differences between before surgery and the 3 months post-surgery follow-up (p < 0.001 and p = 0.002, respectively). Optic nerve head markers demonstrated relative stability, except for changes in avascular complex density, which was 49.2 ± 2.2% before the surgery and decrease to 47.6 ± 1.7% three months after the operation (P-value = 0.005). CONCLUSION: While CCL appears to maintain the integrity of certain ocular structures, alterations in macular perfusion post-CCL suggest potential effects on retinal blood supply. Long-term monitoring is crucial to understand the implications of these changes, particularly in the context of conditions such as diabetes.

Development of a Static Avascular and Dynamic Vascular Human Skin Equivalent Employing Collagen/Keratin Hydrogels.

One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.

Evaluation of the results of contact lens assisted corneal cross-linking treatment in keratoconus patients with thin corneas.

PURPOSE: We aimed to compare the efficacy and safety of accelerated contact lens-assisted cross-linking (CA-CXL) with Lotrafilcon B and Comfilcon A lenses in keratoconus (KC) patients with thin corneas. STUDY DESIGN: Retrospective, single-center study. MATERIALS AND METHODS: We retrospectively included 51 eyes of 39 KC patients with corneal thickness <400µm after epithelial scraping (Epi-off), who underwent accelerated CA-CXL treatment with Lotrafilcon B (n=20) and Comfilcon A (n=31). Uncorrected and corrected distance visual acuity (UDVA and CDVA), manifest refraction values, corneal topographic data and endothelial cell density were recorded at preoperative and postoperative 1st, 3rd and 6th month controls. RESULTS: CDVA in the Comfilcon A group was higher than CDVA before surgery at 6 months postoperatively (p<0.001). When the two lenses were compared, CDVA was found to be significantly higher in the Lotrafilcon B group in the preoperative, postoperative 1st month and 3rd month values, but there was no significant difference between the postoperative 6th month values (p=0.028, p=0.018, p=0.044, p=0.181, respectively). The maximum keratometry (Kmax) value at the 6th month after surgery in the Comfilcon A group was significantly lower than in the Lotrafilcon B group (p=0,009). There was no significant difference between the endothelial cell density values between the groups (p=0.623, p=0.609, p=0.794, p=0.458, respectively). There was no significant difference between the progression, regression, and stability rates of the two groups (p=0.714). CONCLUSIONS: Accelerated CA-CXL with Lotrafilcon B and Comfilcon A silicone hydrogel lenses is a safe and effective method to stop progression in patients with thin corneas.

Role of the mitochondrial protein cyclophilin D in skin wound healing and collagen secretion.

Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation has been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired reepithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, while its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif-KO mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.

F127-SE-tLAP thermosensitive hydrogel alleviates bleomycin-induced skin fibrosis via TGF-ß/Smad pathway.

BACKGROUND: Skin fibrosis affects the normal function of the skin. TGF-ß1 is a key cytokine that affects organ fibrosis. The latency-associated peptide (LAP) is essential for TGF-ß1 activation. We previously constructed and prepared truncated LAP (tLAP), and confirmed that tLAP inhibited liver fibrosis by affecting TGF-ß1. SPACE peptide has both transdermal and transmembrane functions. SPACE promotes the delivery of macromolecules through the stratum corneum into the dermis. This study aimed to alleviate skin fibrosis through the delivery of tLAP by SPACE. METHODS: The SPACE-tLAP (SE-tLAP) recombinant plasmid was constructed. SE-tLAP was purified by nickel affinity chromatography. The effects of SE-tLAP on the proliferation, migration, and expression of fibrosis-related and inflammatory factors were evaluated in TGF-ß1-induced NIH-3T3 cells. F127-SE-tLAP hydrogel was constructed by using F127 as a carrier to load SE-tLAP polypeptide. The degradation, drug release, and biocompatibility of F127-SE-tLAP were evaluated. Bleomycin was used to induce skin fibrosis in mice. HE, Masson, and immunohistochemistry were used to observe the skin histological characteristics. RESULTS: SE-tLAP inhibited the proliferation, migration, and expression of fibrosis-related and inflammatory factors in NIH-3T3 cells. F127-SE-tLAP significantly reduced ECM production, collagen deposition, and fibrotic pathological changes, thereby alleviating skin fibrosis. CONCLUSION: F127-SE-tLAP could increase the transdermal delivery of LAP, reduce the production and deposition of ECM, inhibit the formation of dermal collagen fibers, and alleviate the progression of skin fibrosis. It may provide a new idea for the therapy of skin fibrosis.

Comparative evaluation of biomechanical changes and aberration profile following accelerated collagen cross-linking using hypo-osmolar and iso-osmolar riboflavin: A prospective study.

PURPOSE: To compare the changes encountered in corneal biomechanics and aberration profile following accelerated corneal collagen cross-linking (CXL) using hypo-osmolar and iso-osmolar riboflavin in corneal thicknesses of <400 and >400 microns, respectively. METHODS: This is a prospective, interventional, comparative study involving 100 eyes of 75 patients with progressive keratoconus. Eyes were divided into two groups based on corneal thickness: group 1 included eyes with a corneal thickness of <400 microns who underwent hypo-osmolar CXL, and group 2 included eyes with a corneal thickness of >400 microns who underwent iso-osmolar CXL. Corneal biomechanical and aberration profiles were evaluated and compared between groups. RESULTS: In group 1, all higher-order aberrations (HOA) except secondary astigmatism significantly decreased from baseline; however, in group 2, only coma and trefoil decreased. The corneal resistance factor and corneal hysteresis significantly improved in both groups, which was significantly greater in group 2 than in group 1. The change in inverse radius, deformation amplitude, and tomographic biomechanical index was significantly improved in group 2 as compared to group 1. CONCLUSION: Improvement in corrected distance visual acuity and decrease in HOA were significantly better in the hypo-osmolar CXL group; however, the improvement in biomechanical strength of the cornea was significantly better in the iso-osmolar group.

Cell Senescence-Independent Changes of Human Skin Fibroblasts with Age.

Skin ageing is defined, in part, by collagen depletion and fragmentation that leads to a loss of mechanical tension. This is currently believed to reflect, in part, the accumulation of senescent cells. We compared the expression of genes and proteins for components of the extracellular matrix (ECM) as well as their regulators and found that in vitro senescent cells produced more matrix metalloproteinases (MMPs) than proliferating cells from adult and neonatal donors. This was consistent with previous reports of senescent cells contributing to increased matrix degradation with age; however, cells from adult donors proved significantly less capable of producing new collagen than neonatal or senescent cells, and they showed significantly lower myofibroblast activation as determined by the marker α-SMA. Functionally, adult cells also showed slower migration than neonatal cells. We concluded that the increased collagen degradation of aged fibroblasts might reflect senescence, the reduced collagen production likely reflects senescence-independent processes.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-ß (TGFß) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Extracellular Vesicles of Patients on Peritoneal Dialysis Inhibit the TGF-ß- and PDGF-B-Mediated Fibrotic Processes.

Among patients on peritoneal dialysis (PD), 50-80% will develop peritoneal fibrosis, and 0.5-4.4% will develop life-threatening encapsulating peritoneal sclerosis (EPS). Here, we investigated the role of extracellular vesicles (EVs) on the TGF-ß- and PDGF-B-driven processes of peritoneal fibrosis. EVs were isolated from the peritoneal dialysis effluent (PDE) of children receiving continuous ambulatory PD. The impact of PDE-EVs on the epithelial-mesenchymal transition (EMT) and collagen production of the peritoneal mesothelial cells and fibroblasts were investigated in vitro and in vivo in the chlorhexidine digluconate (CG)-induced mice model of peritoneal fibrosis. PDE-EVs showed spherical morphology in the 100 nm size range, and their spectral features, CD63, and annexin positivity were characteristic of EVs. PDE-EVs penetrated into the peritoneal mesothelial cells and fibroblasts and reduced their PDE- or PDGF-B-induced proliferation. Furthermore, PDE-EVs inhibited the PDE- or TGF-ß-induced EMT and collagen production of the investigated cell types. PDE-EVs contributed to the mesothelial layer integrity and decreased the submesothelial thickening of CG-treated mice. We demonstrated that PDE-EVs significantly inhibit the PDGF-B- or TGF-ß-induced fibrotic processes in vitro and in vivo, suggesting that EVs may contribute to new therapeutic strategies to treat peritoneal fibrosis and other fibroproliferative diseases.

De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.