Collagen Fibril Diameter Research Articles

Substitution of murine type I collagen A1 3-hydroxylation site alters matrix structure but does not recapitulate osteogenesis imperfecta bone dysplasia

Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.

Optical Microscopy and Electron Microscopy for the Morphological Evaluation of Tendons: A Mini Review.

The morphological characteristics of tendons have been thoroughly evaluated via microscopy. Optical microscopy and electron microscopy are the most commonly used techniques for tendon tissue observation. According to the principles of both microscopy types, preparation and evaluation methods vary. Simple optical microscopy is commonly used in the observation of cells and extracellular matrix, and many stains, including hematoxylin–eosin, Van Gieson, Prussian blue, Alcian blue, and toluidine blue, are used for evaluating cells, collagen fiber arrangement, and noncollagenous proteins. Histological scoring systems have been used in many studies for semi‐quantification. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most commonly used electron microscopy types, and special consideration is needed for the fixation and embedding protocols. Glutaraldehyde followed by osmium is most commonly used in the chemical fixation of tendon tissue, followed by epoxy resin embedment. Longitudinal sections captured in SEM images show the arrangement of collagen fibrils and the cells and lipid drops among them, while cross sections captured in TEM images show the diameter and distribution of collagen fibrils. SEM and TEM are used together for comprehensive evaluations. This mini review is focused on the preparation methodology and related evaluation indexes for the morphological evaluation of tendons.

Loss of tenomodulin expression is a risk factor for age-related intervertebral disc degeneration.

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti‐angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament‐specific marker and anti‐angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age‐dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd − / −) mice exhibited more rapid progression of age‐related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd − / − Chm1 − / −) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF‐derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age‐related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age‐related IVD degeneration.

Development of Transparent Acellular Dermal Matrix as Tissue-Engineered Stroma Substitute for Central Lamellar Keratoplasty.

PurposeTo improve the transparency of the acellular dermal matrix (ADM) and investigate the optical, mechanical and histologic properties and biocompatibility of transparent ADM (TADM) in lamellar keratoplasty.MethodsA stepwise sectioning strategy was applied to determine the transparency distribution of the ADM, and TADM was fabricated accordingly. Transmittance measurements, uniaxial tension testing, and histologic staining were applied to detect its properties. Lamellar keratoplasty was performed in rabbits with TADM, and postoperative evaluations were conducted including the transmittance of the transplant area and histologic staining.ResultsThe transmittance of the ADM increased with increasing depth, and TADM was isolated mechanically at the deepest level. There was a significant improvement in the transmittance of the TADM compared with the ADM, and no significant difference in transmittance between dehydrated TADM and cornea was observed. The elastic modulus of TADM was significantly stronger than that of normal cornea (P = 0.004). TADM consisted of dense collagen fibrils, mainly collagen type I, and the collagen fibril diameter and interfibrillar spacing were determined to be larger than corneal stroma. After lamellar keratoplasty in rabbits, the TADM was well integrated with the host cornea, and transparent cornea without neovascularization was observed at 6 months. Re-epithelization was completed at 1 month, and keratocyte repopulation and collagen remodeling were observed in the graft 3 and 6 months after surgery.ConclusionsThis study presents the transparency distribution of the ADM and a method for obtaining TADM, which demonstrates ideal transparency, strong mechanical properties, and satisfactory biocompatibility when applied in lamellar keratoplasty.

X-Ray Diffraction Imaging of Corneal Ultrastructure.

X-ray scattering enables the structure of collagen-rich tissues, such as the cornea, to be examined at both the molecular and fibrillar level. The high-intensity X-rays available at synchrotron radiation sources, coupled with minimal sample preparation requirements, facilitates the rapid generation of high-quality X-ray scattering data from corneal tissue at a close-to-physiological state of hydration. Analysis of resulting X-ray scatter patterns allows one to quantify numerous structural parameters relating to the average diameter, lateral arrangement and alignment of collagen fibrils within the cornea, as well as the axial and lateral arrangements of collagen molecules within the fibrils. Here we describe the typical experimental setup and considerations involved in the collection of X-ray scattering data from corneal tissue.

Intratendon delivery of leukocyte-rich platelet-rich plasma at early stage promotes tendon repair in a rabbit Achilles tendinopathy model.

Tendinopathy is a great obstacle in clinical practice due to its poor regenerative capacity. The influence of different stages of tendinopathy on effects of leukocyte-rich platelet-rich plasma (Lr-PRP) has not been elucidated. The aim of this study is to investigate the optimal time point for delivery of Lr-PRP on tendinopathy. A tendinopathy model was established by local collagenase injection on the rabbit Achilles tendon. Then after collagenase induction, following treatments were applied randomly on the lesion: (a) 200 μl of Lr-PRP at 1 week (PRP-1 group), (b) 200 μl of saline at 1 week (Saline-1 group), (c) 200 μl of Lr-PRP at 4 weeks (PRP-2 group), and (d) 200 μl of saline at 4 weeks (Saline-2 group). Six weeks after collagenase induction, outcomes were assessed by magnetic resonance imaging, cytokine quantification, gene expression, histology, and transmission electron microscopy. Our results demonstrated that PRP-1 group had the least cross-sectional area and lesion percent of the involved tendon, as well as the lowest signal intensity in magnetic resonance imaging among all groups. However, the PRP-2 group showed larger cross-sectional area than saline groups. Enzyme-linked immunosorbent assay indicated that PRP-1 group had a higher level of interleukin-10 but lower level of interleukin-6 when compared with PRP-2 and saline groups. Meanwhile, the highest expression of collagen (Col) 1 in PRP-1 and Col 3, matrix metalloproteinase (MMP)-1, and MMP-3 in PRP-2 was found. Histologically, the PRP-1 showed better general scores than PRP-2, and no significant difference was found between the PRP-2 and saline groups. For transmission electron microscopy, PRP-1 had the largest mean collagen fibril diameter, and the PRP-2 group showed even smaller mean collagen fibril diameter than saline groups. In conclusion, intratendon delivery of Lr-PRP at early stage showed beneficial effect for repair of tendinopathy but not at late stage. For translation of our results to clinical circumstances, further studies are still needed.

Age-related changes of tendon fibril micro-morphology and gene expression.

Aging is hypothesized to be associated with changes in tendon matrix composition which may lead to alteration of tendon material properties and hence propensity to injury. Altered gene expression may offer insights into disease pathophysiology and thus open new perspectives toward designing pathophysiology‐driven therapeutics. Therefore, the current study aimed at identifying naturally occurring differences in tendon micro‐morphology and gene expression of newborn, young and old horses. Age‐related differences in the distribution pattern of tendon fibril thickness and in the expression of the tendon relevant genes collagen type 1 (Col1), Col3, Col5, tenascin‐C, decorin, tenomodulin, versican, scleraxis and cartilage oligomeric matrix protein were investigated. A qualitative and quantitative gene expression and collagen fibril diameter analysis was performed for the most frequently injured equine tendon, the superficial digital flexor tendon, in comparison with the deep digital flexor tendon. Most analyzed genes (Col1, Col3, Col5, tenascin‐C, tenomodulin, scleraxis) were expressed at a higher level in foals (age ≤ 6 months) than in horses of 2.75 years (age at which flexor tendons become mature in structure) and older, decorin expression increased with age. Decorin was previously reported to inhibit the lateral fusion of collagen fibrils, causing a thinner fibril diameter with increased decorin concentration. The results of this study suggested that reduction of tendon fibril diameters commonly seen in equine tendons with increasing age might be a natural age‐related phenomenon leading to greater fibril surface areas with increased fibrillar interaction and reduced sliding at the fascicular/fibrillar interface and hence a stiffer interfascicular/interfibrillar matrix. This may be a potential reason for the higher propensity to tendinopathies with increasing age.

Phenotypic Properties of Collagen in Dentinogenesis Imperfecta Associated with Osteogenesis Imperfecta.

IntroductionDentinogenesis imperfecta type 1 (OIDI) is considered a relatively rare genetic disorder (1:5000 to 1:45,000) associated with osteogenesis imperfecta. OIDI impacts the formation of collagen fibrils in dentin, leading to morphological and structural changes that affect the strength and appearance of teeth. However, there is still a lack of understanding regarding the nanoscale characterization of the disease, in terms of collagen ultrastructure and mechanical properties. Therefore, this research presents a qualitative and quantitative report into the phenotype and characterization of OIDI in dentin, by using a combination of imaging, nanomechanical approaches.MethodsFor this study, 8 primary molars from OIDI patients and 8 primary control molars were collected, embedded in acrylic resin and cut into longitudinal sections. Sections were then demineralized in 37% phosphoric acid using a protocol developed in-house. Initial experiments demonstrated the effectiveness of the demineralization protocol, as the ATR-FTIR spectral fingerprints showed an increase in the amide bands together with a decrease in phosphate content. Structural and mechanical analyses were performed directly on both the mineralized and demineralized samples using a combination of scanning electron microscopy, atomic force microscopy, and Wallace indentation.ResultsMesoscale imaging showed alterations in dentinal tubule morphology in OIDI patients, with a reduced number of tubules and a decreased tubule diameter compared to healthy controls. Nanoscale collagen ultrastructure presented a similar D-banding periodicity between OIDI and controls. Reduced collagen fibrils diameter was also recorded for the OIDI group. The hardness of the (mineralized) control dentin was found to be significantly higher (p<0.05) than that of the OIDI (mineralized) dentine. Both the exposed peri- and intratubular dentinal collagen presented bimodal elastic behaviors (Young’s moduli). The control samples presented a stiffening of the intratubular collagen when compared to the peritubular collagen. In case of the OIDI, this stiffening in the collagen between peri- and intratubular dentinal collagen was not observed and the exposed collagen presented overall a lower elasticity than the control samples.ConclusionThis study presents a systematic approach to the characterization of collagen structure and properties in OIDI as diagnosed in dentin. Structural markers for OIDI at the mesoscale and nanoscale were found and correlated with an observed lack of increased elastic moduli of the collagen fibrils in the intratubular OIDI dentin. These findings offer an explanation of how structural changes in the dentin could be responsible for the failure of some adhesive restorative materials as observed in patients affected by OIDI.

Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - Ultrastructure and 3D transmission electron tomography

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet’s membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

Tendon ECM modified bioactive electrospun fibers promote MSC tenogenic differentiation and tendon regeneration

Proper niche environment is the key factor for mesenchymal stem cells (MSCs) involved tendon-lineage differentiation and regeneration. In the present study, soluble tendon derived extracellular matrix (sTECM) was used to modify aligned electrospun fibers with acid neutralizing effect in order to create an optimal niche environment for tendon regeneration, and its efficacy was evaluated using mouse MSC tenogenic differentiation in vitro and rat Achilles tendon regeneration in vivo. The results showed that sTECM modified ultrafine fibers were more cytocompatible and capable of inducing a predominant tenogenic phenotype of mouse MSCs with the enhanced expression of tendon markers including scleraxis, tenomodulin, collagen III, mohawk homeobox, decorin, fibromodulin and biglycan. By contrast, pure sTECM components led to enhanced multi-lineage differentiation of mouse MSCs including tenogenic lineage. This inductive effect was further enhanced when unilateral mechanical loading was applied in vivo. Moreover, the implantation of sTECM modified scaffold created an ideal niche environment including bioactive sTECM components, topological and mechanical signals, upon in vivo implantation for in situ tendon regeneration. As the results, sTECM modified scaffold in comparison with the non-modified one regenerated higher quality tendon tissue with the features of more mature tissue structure, better tissue organization, cell density and alignment as well as better collagen ultrastructure. Quantitative analysis demonstrated significantly bigger collagen fibril diameter, stronger mechanical property and better tissue grading score and higher expression levels of tenogenic markers when compared to those of control scaffold (p < 0.05). This study demonstrated that sTECM modified pH-neutral ultrafine fibers may represent a novel bioactive scaffold for in situ tendon regeneration, which deserves further investigation in large animals.

A novel ADAMTS17 variant that causes Weill-Marchesani syndrome 4 alters fibrillin-1 and collagen type I deposition in the extracellular matrix

Weill-Marchesani syndrome (WMS) is a rare genetic disorder that affects the musculoskeletal system, the eye, and the cardiovascular system. Individuals with WMS present with short stature, joint contractures, thick skin, microspherophakia, small and dislocated lenses, and cardiac valve anomalies. WMS can be caused by recessive mutations in ADAMTS10 (WMS 1), ADAMTS17 (WMS 4), or LTBP2 (WMS 3), or by dominant mutations in fibrillin-1 (FBN1) (WMS 2); all genes encode secreted extracellular matrix (ECM) proteins. Individuals with WMS 4 due to ADAMTS17 mutations appear to have less severe cardiac involvement and present predominantly with the musculoskeletal and ocular features of WMS. ADAMTS17 is a member of the ADAMTS family of secreted proteases and directly binds to fibrillins. Here we report a novel pathogenic variant in ADAMTS17 that causes WMS 4 in an individual with short stature, brachydactyly, and small, spherical, and dislocated lenses. We provide biochemical and cell biological insights in the pathomechanisms of WMS 4, which also suggest potential biological functions for ADAMTS17. We show that the variant in ADAMTS17 prevents its secretion and we found intracellular accumulation of fibrillin-1 and collagen type I in patient-derived skin fibroblasts. In accordance, transmission electron microscopy revealed elastic fiber abnormalities, decreased collagen fibril diameters, and intracellular collagen accumulation in the dermis of the proband. Together, the data indicate a possible role for ADAMTS17 in the secretion of fibrillin-1 and collagen type I or in their early assembly in the pericellular matrix or the ECM.

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1+/− mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.

Identification of Two Independent COL5A1 Variants in Dogs with Ehlers-Danlos Syndrome.

The Ehlers–Danlos syndromes (EDS) are a heterogeneous group of heritable disorders affecting connective tissues. The mutations causing the various forms of EDS in humans are well characterized, but the genetic mutations causing EDS-like clinical pathology in dogs are not known, thus hampering accurate clinical diagnosis. Clinical analysis of two independent cases of skin hyperextensibility and fragility, one with pronounced joint hypermobility was suggestive of EDS. Whole-genome sequencing revealed de novo mutations of COL5A1 in both cases, confirming the diagnosis of the classical form of EDS. The heterozygous COL5A1 p.Gly1013ValfsTer260 mutation characterized in case 1 introduced a premature termination codon and would be expected to result in α1(V) mRNA nonsense-mediated mRNA decay and collagen V haploinsufficiency. While mRNA was not available from this dog, ultrastructural analysis of the dermis demonstrated variability in collagen fibril diameter and the presence of collagen aggregates, termed ‘collagen cauliflowers’, consistent with COL5A1 mutations underlying classical EDS. In the second case, DNA sequencing demonstrated a p.Gly1571Arg missense variant in the COL5A1 gene. While samples were not available for further analysis, such a glycine substitution would be expected to destabilize the strict molecular structure of the collagen V triple helix and thus affect protein stability and/or integration of the mutant collagen into the collagen V/collagen I heterotypic dermal fibrils. This is the first report of genetic variants in the COL5A1 gene causing the clinical presentation of EDS in dogs. These data provided further evidence of the important role of collagen V in dermal collagen fibrillogenesis. Importantly, from the clinical perspective, we showed the utility of DNA sequencing, combined with the established clinical criteria, in the accurate diagnosis of EDS in dogs.

The collagen metabolism affects the scleral mechanical properties in the different processes of scleral remodeling

PurposeTo increase the understanding of collagen metabolism as related to scleral remodeling in the process of emmetropization and myopization. MethodsAn eyelid suture that lasted 60 days was performed on one-month-old rabbits to establish an experimental monocular visual deprivation myopia model and the axial length was recorded. HE staining and transmission electron microscope were used to observe the scleral structure. The Instron 5544 was used to measure scleral elastic modulus after measuring the scleral thickness. The content of scleral tissue collagen was determined by detecting the hydroxyproline quantity and the collagen I α1, MMP-2, and TIMP-2 mRNA expression were detected by RT-PCR Kit. ResultsDuring regular emmetropization, the scleral collagen synthesis was higher than decomposition and the diameter of collagen fibrils increased, and then the sclera thickened and mechanical properties improved. During myopization, the scleral collagen decomposition was higher than synthesis and the diameter of collagen fibrils was smaller, and then the sclera thinner and mechanical properties lower, which resulted in a difference in scleral tissue strain. ConclusionThe results suggested that there were different scleral remodeling process between the emmetropization and myopization, and the collagen metabolism affected the scleral mechanical properties and scleral remodeling and then affected the scleral growth, axial elongation, and even myopization.

Abstract 61: ECM scaffolds to elucidate the role of Col11a1 in head and neck cancer progression

Abstract Cancer progression is linked to physical properties, cellular and biochemical composition of the tissue environment including the extracellular matrix (ECM). The ECM not only provides signaling cues by presenting ligands, but also plays a key role in mechano-sensing: modulation of biochemical pathways by physical forces. Collagens are one of the most abundant components of the ECM. We previously reported that head and neck squamous cell carcinoma (HNSCC), which is the 6th most common cause of cancer mortality worldwide with &amp;lt; 40% 5-year survival rates, express higher levels of collagen 11a1 (Col11a1) than normal adjacent mucosa. Col11a1 is a minor fibrillar collagen whose main physiologic role is to regulate the diameter of major collagen fibrils in the cartilage. Although reported that Col11a1 facilitates HNSCC proliferation and invasion, not much is known about its role in the tumor microenvironment. We tested the hypothesis that Col11a1-cancer interdependence facilitates tumor adhesion and metastatic colonization. We developed ECM substrates from cell lines and HNSCC tumor tissue that were characterized by scanning electron microscopy and immunofluorescence microscopy. Electrospun meshes with ECM substrates were developed and used to assess HNSCC adhesion, proliferation and motility. We used a 3D multi-cell type spheroid culture model called the primitive-lung-in-a-dish (PLiD) to demonstrate that Col11a1 siRNA targeting attenuates HNSCC colonization in the lung. Further, using homogenized and lyophilized decellularized ECM from mouse lungs, we demonstrate that Col11a1 regulates motility of HNSCC. Overall, we have used several models to elucidate the role of Col11a1 in HNSCC adhesion and metastatic colonization. Citation Format: Sufi M. Thomas, Jonathan Enders, Greta Isai, Andras Czirok. ECM scaffolds to elucidate the role of Col11a1 in head and neck cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 61.

A sclerocornea-associated RAD21 variant induces corneal stroma disorganization

Sclerocornea is a cornea opacification disorder. Disorganized corneal stroma fibrils are observed in patients’ cornea. Previously we identified a RAD21C1348T variant that is associated with a peripheral sclerocornea pedigree. To explore whether this RAD21 variant can induce sclerocornea-related phenotype, and to investigate the possible mechanisms of such phenotype, the orthologous rad21 wild-type and variant mRNAs were injected into Xenopus laevis embryos and the developed eyes were subjected for histological examination. Transmission electron microscopy was applied for corneal stroma organization check. rad21 is highly expressed in the eye region during X. laevis development. Disrupted eye development was observed in the rad21 variant injected embryos. Disorganized corneal stroma and decreased diameters of collagen fibrils were observed in the rad21 variant injected X. laevis eyes. These eye defects can be rescued by overexpression of the wild-type rad21. Histological examination found stroma attracting center, a key structure in X. laevis corneal development, was impaired in rad21 variant injected embryos. Our results suggest a key role of RAD21 during corneal development. Our data indicates the RAD21R450C variant contributes to peripheral sclerocornea by disturbing collagen fibril organization in the corneal stroma.

Compressed collagen intermixed with cornea-derived decellularized extracellular matrix providing mechanical and biochemical niches for corneal stroma analogue

Compressed collagen is a promising scaffold for corneal stroma analogue due to its facile incorporation of keratocytes while mimicking the mechanical niche of a native cornea with dense collagen fibrillar structures. However, it does not offer the sufficient biochemical niche crucial for in vivo-like quiescent keratocyte phenotype. In this study, we engineered a scaffold for a corneal stroma analogue that mimics both the mechanical and biochemical niches of the corneal stroma by introducing cornea-derived decellularized extracellular matrix (Co-dECM) to the collagen compression process. The compressed collagen intermixed with Co-dECM (COLEM; Co-dECM content, <50 wt%) maintained a uniform structure and showed an elastic modulus and tensile strength on the order of 100 kPa, which is comparable with that of conventional compressed collagen. The COLEM with the 50 wt% Co-dECM content was found to possess 2-fold higher amount of the glycosaminoglycans as compared to the compressed collagen. The biochemical components of Co-dECM in the COLEM were verified to significantly promote the expression of quiescent keratocyte-specific genes, i.e., KERA and ALDH3A1, while improving the optical transmittance of the COLEM by reducing the diameter of collagen fibrils. The ability of the COLEM to construct multicellular in vitro corneal tissue was demonstrated by an additional corneal epithelial cell culture. The results support the hypothesis that COLEM has strong potential use in the development of corneal equivalent for in vitro models and tissue transplantation.

Effects of scleral collagen crosslinking with different carbohydrate on chemical bond and ultrastructure of rabbit sclera: Future treatment for myopia progression.

BackgroundMyopia is the most common ocular disorder and is mainly caused by axial elongation of the sclera. If the stiffness of sclera increased, it can inhibit myopia progression. The aim of this study is to compare the effect of the collagen crosslinking with different types and concentrations of carbohydrates on chemical bond and ultrastructural change of rabbit sclera.MethodsNine New Zealand white rabbits were treated with five, sequential sub-Tenon injections of 0.15 mL solutions of ribose, sucrose, and glycogen of 0.1, 0.2 and 0.4 M concentration at the right eye over 14 days. Ten weeks after the last injection, the rabbits were sacrificed and chemical bond and ultrastructural changes were compared with those of the untreated left sclera using Raman spectroscopy, atomic force microscopy (AFM), and histology.ResultsRaman spectroscopy of the control and cross-linked rabbit sclera tissue revealed different types of collagen interactions. Raman shift of 919 cm-1 (C-C stretching and vibration of the proline ring in collagen) was the highest in ribose, followed by sucrose and glycogen. Total energy intensity was also highest in ribose, followed by sucrose and glycogen, and showed a tendency to increase at higher concentrations. AFM revealed interlocking arrangements of collagen fibrils. The collagen fibril diameter was 105.6 ± 21.2 nm, 109.4 ± 28.8 nm, 113.1 ± 30.8 nm and 137.6 ± 25.3 nm for control group, 0.4 M glycogen, sucrose, and ribose, respectively. Histology indicated increased density of the collagen bundle and no increase in inflammatory cell recruitment compared to control at high concentrations of ribose.ConclusionsScleral crosslinking using glycation increased the scleral biomechanical rigidity and these results were particularly pronounced in ribose. Scleral crosslinking using glycation may be a promising method for inhibiting high myopia progression.

Mechanical Factor Analysis on the Sclera: Changes of Scleral Mechanical Properties in the Process of Emmetropization and Myopization and the Effects of Mechanical Stimulation on Scleral Remodeling

To increase the understanding of mechanical factors as related to scleral remodeling in the process of emmetropization and myopization. An eyelid suture that lasted 60 days was performed on one-month-old rabbits to establish an experimental monocular visual deprivation myopia model and the axial length was recorded. HE staining and transmission electron microscope were used to observe the scleral structure. The Instron 5544 was used to measure scleral elastic modulus after measuring the scleral thickness. The scleral fibroblasts were isolated and were cultured under mechanical stimulation using the FX-4000 system. The collagen expression and collagen I α1, MMP-2, and TIMP-2 mRNA expression were detected. During regular emmetropization, the scleral collagen synthesis was higher than decomposition and the diameter of collagen fibrils increased, and then the sclera thickened and mechanical properties improved. During myopization, the scleral collagen decomposition was higher than synthesis and the diameter of collagen fibrils was smaller, and then the sclera thinner and mechanical properties lower, which resulted in a difference in scleral tissue strain. The proper (smaller) mechanical stimulation could improve the collagen synthesis and reduce the decomposition. However, the larger mechanical stimulation could reduce the collagen synthesis and enhance the decomposition. The results suggested that the scleral structure, collagen metabolism, and mechanical properties changed in the process of emmetropization and myopization, and mechanical stimulation affected the scleral remodeling. In addition, the changes in mechanical factors were an important cause of scleral growth, axial elongation, and even myopization. Funding Statement: This study was supported by the National Natural Science Foundation of China (11802209), the Natural Science Foundation of Shandong Province China (ZR2019MA005). Declaration of Interests: The authors have no proprietary interest in any of the materials discussed in this article. Ethics Approval Statement: The experimental protocols were approved by the Animal Care and Protection Committee of Weifang Medical University. We confirm that all experiments were performed in accordance with relevant guidelines and regulations.

Building physiologically‐relevant models of developing cartilage

Perlecan, or heparan sulfate proteoglycan (Hspg2), is an extracellular matrix (ECM) protein localized to the pericellular matrix (PCM). When Hspg2 is knocked down, the phenotype mimics the skeletal defects observed in human Schwartz‐Jampel syndrome. We previously demonstrated, using atomic force microscopy, that perlecan knockdown significantly decreased the stiffness of the ECM and chondrocytes in developing cartilage; however, it is not clear what changes occur in ECM structure and organization to cause this decrease. To address this question, our goal is to develop computational models that incorporate geometries and material properties that will enable us to analyze the mechanical response of cells in situ. Therefore, we created 3D physiologically‐derived geometries that can generate repeatable simulations of cartilage mechanics. In parallel, we are using mass spectrometry (LC‐MS/MS) and transmission electron microscopy (TEM) to define how cartilage composition and organization changes as a function of development and perlecan knockdown.Multi‐cellular geometries from embryonic day (E)16.5 and postnatal day (P)3 murine cartilage were imaged in 3D using confocal microscopy. Image stacks were processed using MATLAB to create multicellular geometries for finite element analysis using ANSYS (Fig 1A,B). Geometries based on confocal images and isolated, single cell models were compressed 5% and cells and ECM strain were compared (Fig 1C,D). Our simulations indicated that cells had similar strains at both time points, even though cell and ECM stiffness at P3 was significantly higher than at E16.5. In addition, the ECM at P3 took on more strain than at E16.5. The isolated, single cell geometries underestimated both cell and ECM strain and were not able to capture the similarity in cell strain observed with physiologically‐derived geometries.A limitation of the current model is that cells and ECM were treated as homogenous materials, whereas native cartilage is a biphasic material comprised of type II collagen fibrils embedded in an amorphous proteoglycan matrix. To identify how ECM composition and organization changes, cartilage from distal humeri or femurs were dissected from E16.5 and P3 wildtype, heterozygous, and homozygous Hspg2 mice for analysis via LC‐MS/MS or TEM. Proteins were extracted with guanidine‐HCl and processed for LC‐MS/MS. Relative increases in the abundance of ECM and associated proteins, including collagen types II &amp; IX and aggrecan highlighted expected developmental changes between E16.5 and P3. Multiple proteins were significantly more abundant in homozygous vs. wildtype P3 mice, including collagens type II &amp; IX, fibronectin and matrilin‐3. Surprisingly, TEM studies indicate that collagen fibril density and diameter decreased between wildtype and homozygous mice, suggesting that perlecan knockdown leads to a decrease in cross‐linking and increased ECM extractability. We aim to integrate these changes in material properties with our simulations to generate a computational model that can describe and predict the mechanics of developing cartilage.Support or Funding InformationThis work was supported by the National Institutes of Health [R21 AR069248, R01 AR071359 and DP2 AT009833 to S.C.].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.