Distinctive structure, composition and biomechanics of collagen fibrils in vaginal wall connective tissues associated with pelvic organ prolapse.

Collagen is the predominant structural protein within connective tissues. Pelvic organ prolapse (POP) is characterized by weakening of the pelvic floor connective tissues and loss of support for pelvic organs. In this study, we examined the multiscale structure, molecular composition and biomechanics of native collagen fibrils in connective tissues of the posterior vaginal fornix collected from healthy women and POP patients, and established the correlation of these properties with clinical POP quantification (POP-Q) scores. The collagen characteristics, including collagen amount, ratio of Collagen I and Collagen III, collagen fibril d-period, alignment and stiffness, were found to change progressively with the increase of the clinical measurement of Point C, a measure of uterine descent and apical prolapse. The results imply that a severe prolapse is associated with stiffer collagen fibrils, reduced collagen d-period, increased fibril alignment and imbalanced collagen synthesis, degradation and deposition. Additionally, prolapse progression appears to be synchronized with deterioration of the collagen matrix, suggesting that a POP-Q score obtained via a non-invasive clinical test can be potentially used to quantitatively assess collagen abnormality of a patient's local tissue. STATEMENT OF SIGNIFICANCE: Abnormal collagen metabolism and deposition are known to associate with connective tissue disorders, such as pelvic organ prolapse. Quantitative correlation of the biochemical and biophysical characteristics of collagen in a prolapse patient's tissue with the clinical diagnostic measurements is unexplored and unestablished. This study fills the knowledge gap between clinical prolapse quantification and the individual's cellular and molecular disorders leading to connective tissue failure, thus, provides the basis for clinicians to employ personalized treatment that can best manage the patient's condition and to alert pre-symptomatic patients for early management to avoid unwanted surgery.

Distinctive roles of fibrillar collagen I and collagen III in mediating fibroblast-matrix interaction: A nanoscopic study.

One major goal in tissue engineering is to create functional materials, mimicking scaffolds in native tissues, to modulate cell function for tissue repair. Collagen is the most abundant structural protein in human body. Though collagen I (COLI) and collagen III (COLIII) are the predominant collagen types in connective tissues and they form stable hybrid fibrils at varied ratios, cell responses to the hybrid matrices are underinvestigated. In this work, we aim to explicate the distinctive roles of COLI and COLIII in fibroblast activation. Unidirectionally aligned COLI, COLIII and COLI-COLIII hybrid nanofibrils were generated via epitaxial growth of collagen on mica. AFM analyses revealed that, with the increase of COLI/COLIII ratio, the fibril width and stiffness increased and the binding affinity of cells to the matrix decreased. A hybrid matrix was found to activate fibroblasts the most effectively, characterized by extensive cell polarization with rigid stress fiber bundles and high α-SMA expression, and by the highest-level of collagen synthesis. It is ascribed to the fine balance between biochemical and biophysical cues achieved on the hybrid matrix. Thus, matrices of aligned COLI-COLIII hybrid fibrils and their derived multifunctional composites can be good candidates of implantation scaffolds for tissue regeneration.

Aligned Collagen-CNT Nanofibrils and the Modulation Effect on Ovarian Cancer Cells.

Fibrillar collagen is a one-dimensional biopolymer and is the most abundant structural protein in the extracellular matrix (ECM) of connective tissues. Due to the unique properties of carbon nanotubes (CNTs), considerable attention has been given to the application of CNTs in developing biocomposite materials for tissue engineering and drug delivery. When introduced to tissues, CNTs inevitably interact and integrate with collagen and impose a discernible effect on cells in the vicinity. The positive effect of the collagen-CNT (COL-CNT) matrix in tissue regeneration and the cytotoxicity of free CNTs have been investigated extensively. In this study, we aimed to examine the effect of COL-CNT on mediating the interaction between the matrix and SKOV3 ovarian cancer cells. We generated unidirectionally aligned collagen and COL-CNT nanofibrils, mimicking the structure and dimension of collagen fibrils in native tissues. AFM analysis revealed that the one-dimensional structure, high stiffness, and low adhesion of COL-CNT greatly facilitated the polarization of SKOV3 cells by regulating the ß-1 integrin-mediated cell-matrix interaction, cytoskeleton rearrangement, and cell migration. Protein and gene level analyses implied that both collagen and COL-CNT matrices induced the epithelial-mesenchymal transition (EMT), and the COL-CNT matrix prompted a higher level of cell transformation. However, the induced cells expressed CD44 at a reduced level and MMP2 at an increased level, and they were responsive to the chemotherapy drug gemcitabine. The results suggested that the COL-CNT matrix induced the transdifferentiation of the epithelial cancer cells to mature, less aggressive, and less potent cells, which are inapt for tumor metastasis and chemoresistance. Thus, the presence of CNT in a collagen matrix is unlikely to cause an adverse effect on cancer patients if a controlled dose of CNT is used for drug delivery or tissue regeneration.

Silk-CNT Mediated Fibroblast Stimulation toward Chronic Wound Repair.

BACKGROUND: Diabetic patients suffer from chronic wounds partly due to altered function of fibroblasts. Fibroblasts of diabetic patients synthesize collagen I (COLI) at a much higher level than collagen III (COLIII), resulting in delayed tissue granulation and, consequently, a delay in the overall wound healing process. METHODS: We aimed to revive the matrix protein productivity of diabetic fibroblasts by employing aligned, electrically conductive and biocompatible spider silk-CNT fibers as a cell culture matrix to mediate the electrical stimulation of fibroblasts to induce cell polarization and activation. RESULTS: A 5.2 and 42.7 fold increase in COLI and COLIII production was induced in diabetic fibroblasts. The stimulated cells synthesized a substantially high level of COLIII to reduce the abnormally high COLI/COLIII ratio, and the matrix metalloproteinases expression was markedly suppressed. The protein expression profile was consistent with favorable wound healing. The modulation effect was also demonstrated in normal fibroblasts of healthy individuals, suggesting that the developed method can be utilized generally for connective tissue repair. Silkworm silk-CNT fibers corroborated similar effects on restoring the function of diabetic fibroblasts. CONCLUSIONS: The approach of using an engineered biopolymer matrix to remedy dysfunctional fibroblasts of patients offers the opportunity of developing personalized cell therapy for noninvasive treatments and inspires the design of multi-functional biometrics for effective tissue regeneration.

Electrospun protein-CNT composite fibers and the application in fibroblast stimulation.

Functional biopolymer scaffolds are in high demand for tissue regeneration. In this study, we incorporated functionalized CNT in collagen or silk protein solution to generate biocomposite fibers by electrospinning. The addition of CNT reinforced the strength of the scaffolds and rendered the fibers electrical conductivity to not only facilitate the E-spun fiber formation but also grant the fibers an additional functionality that can be utilized for cell stimulation. Considering fiber dimension, alignment, mechanical strength, electrical conductivity and biocompatibility, silk-CNT fibers containing a minute amount of CNT (0.05%) outperformed other fiber types. The modulation effect of these fibers was examined by their application in inducing polarization and activation of fibroblasts with cellular deficit. While the fibroblasts on both collagen-CNT and silk-CNT fibers synthesized a substantially higher level of collagen type III (COLIII) than cells on pure protein fibers to reduce the abnormally high COLI/COLIII ratio, electrical stimulation boosted the collagen productivity by 20 folds in cells on silk-CNT than on collagen-CNT due to silk-CNT's high electrical conductivity. The developed approach can be potentially utilized to remedy the dysfunctional fibroblasts for therapeutic treatment of diseases and health conditions associated with collagen disorder.

Optimization of Glutaraldehyde Vapor Treatment for Electrospun Collagen/Silk Tissue Engineering Scaffolds.

Freestanding fibrous matrices with proper protein composition and desirable mechanical properties, stability, and biocompatibility are in high demand for tissue engineering. Electrospun (E-spun) collagen-silk composite fibers are promising tissue engineering scaffolds. However, as-spun fibers are mechanically weak and unstable. In this work, we applied glutaraldehyde (GA) vapor treatment to improve the fiber performance, and the effect on the properties of E-spun collagen-silk fibers was studied systematically. GA treatment was found to affect collagen and silk distinctively. Whereas GA chemically links collagen peptides, it induces conformational transitions to enrich ß-sheets in silk. The combined effects impose a control of the mechanical properties, stability, and degradability of the composite fibers, which are dependent on the extent of GA treatment. In addition, a mild treatment of the fibers did not diminish cell proliferation and viability. However, overly treated fibers demonstrated reduced cell-matrix adhesion. The understanding of GA treatment effects on collagen, silk, and the composite fibers enables effective control and fine tuning of the fiber properties to warrant their diverse in vitro and in vivo applications.

Identifying distinct nanoscopic features of native collagen fibrils towards early diagnosis of pelvic organ prolapse.

Pelvic organ prolapse (POP) is characterized by weakening of the connective tissues and loss of support for the pelvic organs. Collagen is the predominant, load-bearing protein within pelvic floor connective tissues. In this study, we examined the nanoscopic structures and biomechanics of native collagen fibrils in surgical, vaginal wall connective tissues from healthy women and POP patients. Compared to controls, collagen fibrils in POP samples were bulkier, more uneven in width and stiffer with aberrant D-period. Additionally, the ratio of collagen I (COLI) and collagen III (COLIII) is doubled in POP with a concomitant reduction of the amount of total collagen. Thus, POP is characterized by abnormal biochemical composition and biophysical characteristics of collagen fibrils that form a loose and fragile fiber network accountable for the weak load-bearing capability. The study identifies nanoscale alterations in collagen as diagnostic markers that could enable pre-symptomatic or early diagnosis of POP. FROM THE CLINICAL EDITOR: Pelvic organ prolapse (POP) occurs due to abnormalities of the supporting connective tissues. The underlying alterations of collagen fibers in the connective tissues have not been studied extensively. In this article, the authors showed that collagen fibrils in POP patients were much different from normal controls. The findings may provide a framework for the diagnosis of other connective diseases.