Collagen Lamellae Research Articles

Molecular crowding of collagen: A pathway to produce highly-organized collagenous structures

Collagen in vertebrate animals is often arranged in alternating lamellae or in bundles of aligned fibrils which are designed to withstand in vivo mechanical loads. The formation of these organized structures is thought to result from a complex, large-area integration of individual cell motion and locally-controlled synthesis of fibrillar arrays via cell-surface fibripositors (direct matrix printing). The difficulty of reproducing such a process in vitro has prevented tissue engineers from constructing clinically useful load-bearing connective tissue directly from collagen. However, we and others have taken the view that long-range organizational information is potentially encoded into the structure of the collagen molecule itself, allowing the control of fibril organization to extend far from cell (or bounding) surfaces. We here demonstrate a simple, fast, cell-free method capable of producing highly-organized, anistropic collagen fibrillar lamellae de novo which persist over relatively long-distances (tens to hundreds of microns). Our approach to nanoscale organizational control takes advantage of the intrinsic physiochemical properties of collagen molecules by inducing collagen association through molecular crowding and geometric confinement. To mimic biological tissues which comprise planar, aligned collagen lamellae (e.g. cornea, lamellar bone or annulus fibrosus), type I collagen was confined to a thin, planar geometry, concentrated through molecular crowding and polymerized. The resulting fibrillar lamellae show a striking resemblance to native load-bearing lamellae in that the fibrils are small, generally aligned in the plane of the confining space and change direction en masse throughout the thickness of the construct. The process of organizational control is consistent with embryonic development where the bounded planar cell sheets produced by fibroblasts suggest a similar confinement/concentration strategy. Such a simple approach to nanoscale organizational control of structure not only makes de novo tissue engineering a possibility, but also suggests a clearer pathway to organization for fibroblasts than direct matrix printing.

Abnormalities of Stromal Structure in the Bullous Keratopathy Cornea Identified by Second Harmonic Generation Imaging Microscopy

To identify structural alterations in collagen lamellae and the transdifferentiation of keratocytes into myofibroblasts in the corneal stroma of bullous keratopathy (BK) patients and to examine the relation of such changes to the duration of stromal edema or the underlying cause of BK. Six normal human corneas and 16 BK corneas were subjected to second harmonic generation (SHG) imaging microscopy to allow three-dimensional (3-D) reconstruction of collagen lamellae. Expression of α-smooth muscle actin (αSMA) was examined by immunofluorescence analysis and conventional laser confocal microscopy. Collagen lamellae were interwoven at the anterior stroma and uniformly aligned at the posterior stroma, whereas αSMA was not detected throughout the entire stroma of the normal cornea. Nine (56%) and 7 (44%) of the 16 BK corneas showed abnormal collagen structure at the anterior and posterior stroma, respectively. Expression of αSMA was detected in the anterior or posterior stroma of 7 (44%) and 6 (38%) of the 16 BK corneas, respectively. Disorganization of collagen lamellae and myofibroblastic transdifferentiation were detected only in corneas with a duration of stromal edema of at least 12 months. Corneas with BK as a result of birth injury showed abnormal collagen structure at the posterior stroma, whereas those with BK resulting from laser iridotomy did not. Changes in the structure of the entire stroma were detected in BK corneas with a duration of stromal edema of at least 12 months, suggesting that such changes may be progressive. In addition, the underlying cause of BK may influence structural changes at the posterior stroma.

Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants

Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4 h at 37 °C, followed by lyophilization at −10 °C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7 ± 4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7 ± 4.6% and 23.01 ± 3.45 m2/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5 min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.

Collagen-Glycosaminoglycan Coprecipitate and Photochemically Cross-Linked Collagen for Disk Replacement

IntroductionTraditional treatments of severe disk degeneration such as discectomy were not satisfactory. Recent research focuses on developing biological substitutes, using tissue engineering methods, for nucleus pulposus and annulus fibrosus.1,2 A few studies had demonstrated the fabrication of biphasic composite scaffold as nucleus and annulus replacements using alginate and polyglycolic acid and alginate and collagen.3,4 Being the prevalent matrix components in native disc, collagen and glycosaminoglycans (GAGs) are potentially suitable biomaterials for a more nature-simulating scaffold. Previously, we have demonstrated that chemical modifications of collagen by deamination, methylation, and amination were able to control the GAG binding to and retention from the collagen meshwork, resulting in controllable GAG:HYP ratio, while maintaining over 90% cell viability. In this study, the ultrastructure of the coprecipitates fabricated in acidic and neutral pH was evaluated. Moreover, aminated CG coprecipitate, with optimal GAG:HYP ratio, along with mechanically strengthened photochemically crosslinked collagen lamellae previously developed in our lab, were used to fabricate a biphasic composite scaffold. On-going work is underway to test the cytocompatibility and mechanical properties of the composite scaffold. Materials and MethodsFirst, modified collagens (deaminated, aminated, and methylated) were coprecipitated with GAG to form CG coprecipitates, which were observed under SEM before and after neutralization. Second, aminated CG was entrapped as the core of a collagen shell, which was photochemically crosslinked in the presence of rose bengal and argon laser before partial dehydration of the whole biphasic scaffold. Rabbit mesenchymal stem cells would be cultured in the core of the scaffold for 7 days. Scaffolds would be evaluated by immunohistology and cell survival was visualized with live/dead staining. The scaffold would be tested for creep and dynamic responses. ResultsFirst, collagen fibers in modified CGs had distinctive ultrastructural features at different pH. In acidic conditions, fibers were compact with small meshes (∼50 nm) in aminated, methylated, and nontreated CGs. In physiological pH, observations in the three CGs were also distinctive. Meshsize increased in non-treated CG. Sheet-like substance wrapped around the fibers in methylated CG. Granule-like ground substances were found in aminated CG. Seoncd, the feasibility of fabricating a biphasic composite scaffold with aminated CG as the core and the photochemically cross-linked collagen lamellae as the shell has been demonstrated. Preliminary mechanical study demonstrated that a step strain of 72% (pre-strain included) resulted in resistance stress up to 1 MPa, which gradually decreased to 0.5 MPa over 20 minutes. ConclusionThis study demonstrated that there were structural differences between different modified CGs and the fabrication of a biphasic collagen-GAG-based composite scaffold for IVD tissue engineering is feasible. The functional features of the composite scaffold have to be further evaluated.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredSebastineM, Williams DJ. Current developments in tissue engineering of nucleus pulposus for the treatment of intervertebral disk degeneration,” Conference proceedings?: Annual International Conference of the IEEE Engineering in Medicine and Biology Society.IEEE Engineering in Medicine and Biology Society. Conference 2007;2007:6401–6406Nerurkar NL, Baker BM, Sen S, Wible EE, Elliott DM, Mauck RL. Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus. Nature materials 2009;8(12):986–992Mizuno H, Roy AK, Zaporojan V, Vacanti CA, Ueda M, Bonassar LJ. Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs. Biomaterials 2006;27(3):362–370Bowles RD, Williams RM, Zipfel WR, Bonassar LJ. Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disk composite via collagen gel contraction. Tissue engineering. Part A 2010;16(4):1339–1348

Collagen-Glycosaminoglycan Coprecipitate and Photochemically Cross-Linked Collagen for Disk Replacement

Introduction Traditional treatments of severe disk degeneration such as discectomy were not satisfactory. Recent research focuses on developing biological substitutes, using tissue engineering methods, for nucleus pulposus and annulus fibrosus.1,2 A few studies had demonstrated the fabrication of biphasic composite scaffold as nucleus and annulus replacements using alginate and polyglycolic acid and alginate and collagen.3,4 Being the prevalent matrix components in native disc, collagen and glycosaminoglycans (GAGs) are potentially suitable biomaterials for a more nature-simulating scaffold. Previously, we have demonstrated that chemical modifications of collagen by deamination, methylation, and amination were able to control the GAG binding to and retention from the collagen meshwork, resulting in controllable GAG:HYP ratio, while maintaining over 90% cell viability. In this study, the ultrastructure of the coprecipitates fabricated in acidic and neutral pH was evaluated. Moreover, aminated CG coprecipitate, with optimal GAG:HYP ratio, along with mechanically strengthened photochemically crosslinked collagen lamellae previously developed in our lab, were used to fabricate a biphasic composite scaffold. On-going work is underway to test the cytocompatibility and mechanical properties of the composite scaffold. Materials and Methods First, modified collagens (deaminated, aminated, and methylated) were coprecipitated with GAG to form CG coprecipitates, which were observed under SEM before and after neutralization. Second, aminated CG was entrapped as the core of a collagen shell, which was photochemically crosslinked in the presence of rose bengal and argon laser before partial dehydration of the whole biphasic scaffold. Rabbit mesenchymal stem cells would be cultured in the core of the scaffold for 7 days. Scaffolds would be evaluated by immunohistology and cell survival was visualized with live/dead staining. The scaffold would be tested for creep and dynamic responses. Results First, collagen fibers in modified CGs had distinctive ultrastructural features at different pH. In acidic conditions, fibers were compact with small meshes (∼50 nm) in aminated, methylated, and nontreated CGs. In physiological pH, observations in the three CGs were also distinctive. Meshsize increased in non-treated CG. Sheet-like substance wrapped around the fibers in methylated CG. Granule-like ground substances were found in aminated CG. Seoncd, the feasibility of fabricating a biphasic composite scaffold with aminated CG as the core and the photochemically cross-linked collagen lamellae as the shell has been demonstrated. Preliminary mechanical study demonstrated that a step strain of 72% (pre-strain included) resulted in resistance stress up to 1 MPa, which gradually decreased to 0.5 MPa over 20 minutes. Conclusion This study demonstrated that there were structural differences between different modified CGs and the fabrication of a biphasic collagen-GAG-based composite scaffold for IVD tissue engineering is feasible. The functional features of the composite scaffold have to be further evaluated. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared SebastineM, Williams DJ. Current developments in tissue engineering of nucleus pulposus for the treatment of intervertebral disk degeneration,” Conference proceedings?: Annual International Conference of the IEEE Engineering in Medicine and Biology Society.IEEE Engineering in Medicine and Biology Society. Conference 2007;2007:6401–6406 Nerurkar NL, Baker BM, Sen S, Wible EE, Elliott DM, Mauck RL. Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus. Nature materials 2009;8(12):986–992 Mizuno H, Roy AK, Zaporojan V, Vacanti CA, Ueda M, Bonassar LJ. Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs. Biomaterials 2006;27(3):362–370 Bowles RD, Williams RM, Zipfel WR, Bonassar LJ. Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disk composite via collagen gel contraction. Tissue engineering. Part A 2010;16(4):1339–1348

Destructive Radiologic Development of Intravascular Papillary Endothelial Hyperplasia on Skull Bone

Intravascular papillary endothelial hyperplasia (IPEH) is a rare vascular benign lesion that rarely involves the central nervous system with or without skull invasion. We report a rare case of IPEH on the skull bone, which displayed destructive radiologic development associated with hemorrhage. A 14-year-old male presented with an incidentally detected a small enhancing, left frontal osteolytic lesion. Previously, he underwent operation and received adjuvant chemoradiation therapy for cerebellar medulloblastoma. Follow-up magnetic resonance imaging revealed a left frontal bone lesion, which expanded to an approximately 2 cm-sized well-circumscribed osteolytic lesion associated with hemorrhage for 20 months. Frontal craniectomy and cranioplasty were performed. Destructive change was detected on the inner table and diploic space of the skull. The mass had a cystic feature with hemorrhagic content without dural attachment. Pathologic examination showed the capsule consisted of parallel collagen lamellae representing a vascular wall, vascular lumen, which was pathognomonic for IPEH. Immunohistochemical staining revealed that the capsule was positive for CD34 and factor VIII, which favor the final diagnosis of IPEH. This was the first case of intracalvarial IPEH.

Pay attention to the effect of keratocyte

Keratocytes situated between the collagen lamellae in the corneal stroma,are mitotically quiescent cells.The cells can secrete collagens and keratan sulfate proteoglycans which contribute to the transparency of the cornea.Upon injury,keratocytes are stimulated to corrent apoptosis and transite into repair phenotypes,fibroblast and myofibroblast.These repair phenotypes can either promote regeneration or induce fibrotic scar,and the latter is detrimental to the otherwise transparent cornea.Moreover,a population of keratocytes can be induced to appear the stem cell-like characteristics.Therefore,keratocytes are no longer bystanders,but active participants in numerous functions. Key words: Keratocyte; Apoptosis; Fibroblast; Myofibroblast; Stem cell

Characterization of the Cleavage Plane in Descemet's Membrane Endothelial Keratoplasty

To define the cleavage plane between Descemet's membrane (DM) and posterior corneal stroma in Descemet's membrane endothelial keratoplasty (DMEK) concerning its ultrastructural and immunohistochemical characteristics. Observational, consecutive case series. Fifteen corneoscleral buttons from donors 71.5±4.3 years of age stored in Optisol-GS and used for DMEK surgery in 15 consecutive patients. Endothelial cell-DM complexes (EDMs) and corresponding corneoscleral rims were investigated by transmission electron microscopy and immunohistochemistry using a panel of antibodies against adhesive matrix proteins. Ultrastructural and immunohistochemical characteristics of interface and cleavage plane between DM and posterior stroma. Connection between DM and corneal stroma was mediated predominantly by amorphous material of the interfacial matrix and projecting stromal collagen fibers. After DM stripping, the cleavage plane was located consistently between interfacial matrix and posterior stromal collagen lamellae, providing a largely smooth anterior EDM surface exposing the interfacial zone. Interindividual variations in amount and composition of the interfacial matrix resulted in variable degrees of EDM surface irregularities and variable staining patterns for adhesive matrix proteins such as fibronectin, vitronectin, amyloid P, osteonectin/secreted protein acidic and rich in cysteine (SPARC), fibulin-1, fibulin-2, fibulin-3, fibrillin-1, and keratoepithelin. The findings provide evidence for the existence of a physiologic cleavage plane between the interfacial matrix, the anteriormost adhesive zone of DM, and the corneal stroma, suggesting a relatively weak attachment that can be disconnected by mechanical forces. Interindividual variations in structure and composition of the interfacial matrix may provide an explanation for the variable attachment of EDM grafts to the recipients' corneal stroma and thus may affect the postoperative clinical outcome. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Cornea microstructure and mechanical responses measured with nonlinear optical and optical coherence microscopy using sub-10-fs pulses

A combined nonlinear optical microscopy (NLOM) and optical coherence microscopy (OCM) imaging system has been assembled in order to simultaneously capture co-registered volumetric images of corneal morphology and biochemistry. Tracking of cell nuclei visible in the OCM volume enabled the calculation of strain depth profile in response to changes in intraocular pressure for rabbit cornea stroma. Results revealed nonlinear responses with a depth dependent strain distribution, exhibiting smaller strains in the anterior and larger strains in the posterior stroma. Cross-sectional images of collagen lamellae, visible in NLOM, showed inhomogeneous collagen structure along the anterior-posterior direction that correlated well with the noted heterogeneous corneal mechanical responses.

Myofibroblast Metaplasia After Descemet Membrane Endothelial Keratoplasty

To describe myofibroblastic metaplasia of corneal endothelial cells in 2 cases with impaired visual function despite complete graft adherence after Descemet membrane endothelial keratoplasty (DMEK). Interventional case series. In 2 of 90 consecutive DMEK surgeries, the cornea failed to clear up to 6 months postoperatively despite complete graft attachment. After secondary penetrating keratoplasty, both corneal buttons were examined using histopathologic analysis and transmission electron microscopy. Light microscopy revealed distinct corneal endothelial cell attenuation with the presence of an abnormal posterior collagenous layer in both cases. Most of the remaining endothelial cells had an elongated fibroblast-like appearance with immunopositivity for α-smooth muscle actin indicative of myofibroblast metaplasia. Transmission electron microscopy showed a slightly thickened Descemet membrane with an abnormal posterior fibrillar collagenous layer and a myofibroblast-like transformation of the remaining endothelial cells. Descemet membrane grafts closely adjoined the collagen lamellae of the host corneal stroma similar to the Descemet membrane-stroma interface of a normal cornea. Myofibroblastic metaplasia of attenuated corneal endothelial cells with formation of an abnormal posterior collagenous layer may contribute to an impaired visual function despite complete graft adherence after Descemet membrane endothelial keratoplasty (DMEK).

The influence of lamellar orientation on corneal material behavior: biomechanical and structural changes in an avian corneal disorder.

Retinopathy, globe enlarged (RGE) is an inherited genetic disease of chickens with a corneal phenotype characterized by loss of tissue curvature and changes in peripheral collagen fibril alignment. This study aimed to characterize the material behavior of normal and RGE chicken corneas under inflation and compare this with new spatial- and depth-resolved microstructural information to investigate how stromal fibril architecture determines corneal behavior under intraocular pressure (IOP). Six RGE chicken corneas and six age-matched normal controls were tested using trephinate inflation and their stress-strain behavior determined as a function of posterior pressure. Second harmonic generation mulitphoton microscopy was used to compare the in-plane appearance and degree of through-plane interlacing of collagen lamellae between normal and mutant corneas. RGE corneas displayed a 30-130% increase in material stiffness [E(tangent)(RGE) = 0.94 ± 0.18 MPa to 3.09 ± 0.66 MPa; E(tangent)(normals) = 0.72 ± 0.13 MPa to 1.34 ± 0.35 MPa] (P ≤ 0.05). The normal in-plane disposition of anterior collagen in the peripheral cornea was altered in RGE but through-plane lamellar interlacing was unaffected. This article demonstrates changes in corneal material behavior in RGE that are qualitatively consistent with microstructural collagen alterations identified both herein and previously. This study indicates that, in general, changes in stromal fibril orientation may significantly affect corneal material behavior and thereby its response to IOP.

Production of highly aligned collagen lamellae by combining shear force and thin film confinement

Load-bearing tissues owe their mechanical strength to their highly anisotropic collagenous structure. To date attempts to engineer mechanically strong connective tissue have failed, mainly due to a lack of ability to reproduce the native collagen organization in constructs synthesized by cultured cells in vitro. The ability to influence the orientation of self-assembling collagen molecules to produce highly anisotropic structures has applications ranging from de novo engineering of complex tissues to the production of organized scaffolds for cell culture contact guidance. In this investigation we have used the simple technique of spin-coating to produce highly aligned arrays of collagen fibrils. By a simple modification of the method we have also successfully produced orthogonal collagen lamellae. Alternating collagen lamellae are frequently seen in load-bearing tissues such as cornea, annulus fibrosus, and cortical bone. Culturing of corneal fibroblasts on aligned collagen shows that the cells adopt the organization of fibrils. In this investigation we observed the reversal of fibrillar growth direction or “hook” formation similar to that seen previously in a microfluidic shear flow chamber. Although the results of this investigation clearly show that it is possible to produce small areas (1 cm 2) of collagen fibrils with enough alignment to guide fibroblasts, there is evidence that thin film instabilities are likely to be a significant barrier to producing organized collagen fibrils over larger areas. Successful application of this method to produce highly controlled and organized collagenous structures will require the development of techniques to control thin film instability and will be the subject of future work.

Three-Dimensional Analysis of Collagen Lamellae in the Anterior Stroma of the Human Cornea Visualized by Second Harmonic Generation Imaging Microscopy

The structure of collagen lamellae in the anterior stroma of the human cornea is thought to be an important determinant of corneal rigidity. The three-dimensional structure of such collagen lamellae in normal human corneas was examined. The anterior portion of 27 normal human corneas was obtained from donor tissue for Descemet's stripping automated endothelial keratoplasty (DSAEK) surgery, and blocks (∼3-mm square) of the central cornea were examined by second harmonic generation (SHG) imaging microscopy. Each cornea was scanned from the surface of Bowman's layer to a depth of 150 μm, and SHG forward signals were collected. The angles of collagen lamellae immediately below to a depth of 30 μm below Bowman's layer (sutural lamellae) as well as of those at a depth of 50 or 100 μm were measured. The density and width of sutural lamellae were also evaluated. Collagen lamellae in the anterior stroma were evenly distributed and randomly oriented. The angle of sutural lamellae relative to Bowman's layer was 19.19 ± 4.34° (mean ± SD). The angles of collagen lamellae at depths of 50 or 100 μm were 8.91 ± 2.91 and 6.91 ± 2.11°, respectively. The density of sutural lamellae was 910.0 ± 480.4/mm(2), and their width was 13.14 ± 5.03 and 7.11 ± 3.00 μm in the region immediately beneath and 30 μm below Bowman's layer, respectively. Collagen lamellae in the anterior stroma of the normal human cornea are interwoven in three dimensions and adhere densely to Bowman's layer. This structure may contribute to the rigidity and curvature of the anterior portion of the cornea.

The timeline of lamellar basement membrane changes during equine laminitis development

The timing of lamellar basement membrane (BM) changes occurring during laminitis development is incompletely understood. To determine the temporal progression of lamellar BM changes and whether laminin-332 (Ln-332) γ2 cleavage products are generated during laminitis development. Eight clinically normal Standardbred horses were allocated into treatment (n = 5) or sham (n = 3) groups. The treatment group received, via nasogastric intubation, an oligofructose (OF) bolus (10 g/kg bwt) while the sham group was given water. Laminitis induction proceeded for 48 h followed by euthanasia. Lamellar biopsies were obtained prior to dosing and at intervals during the treatment period for analysis (at 12, 18, 24, 30 and 36 h and at 48 h following euthanasia). Changes in lamellar collagen type IV and Ln-332 were first observed at 12 h post dosing. A unique pattern of reactivity for the Ln-332 γ2 antibody D4B5 occurred, in which reactivity was observed only in lamellar tissue affected by laminitis. No bioactive Ln-332 γ2 proteolytic fragments were detected in lamellar samples. Basement membrane changes occurred early during the laminitis process. Direct Ln-332 γ2 cleavage to release biologically active products did not appear to occur. Thus loss of stability or protein interaction of the BM is probably responsible for the γ2 specific reactivity observed. Basement membrane changes may a first step in lamellar failure occurring prior to detection with conventional methods. Thus, more sensitive detection methods of BM changes are required to study laminitis development.

UVA-light and Riboflavin-mediated Corneal Collagen Cross-linking

Keratoconus and other corneal conditions associated with progressive stromal thinning including keratectasia after laser-assisted in situ keratomileusis (LASIK) and pellucid marginal degeneration represent therapeutic challenges. Until recently, available therapies including contact lenses, epikeratoplasty, intrastromal corneal rings, and corneal transplantation targeted, in principle, the abnormal shape of the cornea by mechanical means. The underlying molecular pathogenic mechanisms of corneal ectasia were not addressed with any of these therapies until the introduction of corneal collagen cross-linking to clinical practice in 1999.1 This breakthrough clinical application was the result of multiple laboratory observations and discoveries made over preceding decades. Early experiments of Cannon and Foster2 showed that degraded normal collagen or synthesis of abnormal collagen might play a role in the pathogenesis of keratoconus. This concept was later confirmed by subsequent clinical and experimental studies. Keratoconus was characterized by progressive stromal thinning and ectasia3 as a result of increased expression of lysosomal and proteolytic enzymes4–7 and decreased concentration of protease inhibitors,5,8 which result in corneal thinning and altered configuration of corneal collagen lamellae.9,10

An inverse finite element method for determining the anisotropic properties of the cornea

An inverse finite element method was developed to determine the anisotropic properties of bovine cornea from an in vitro inflation experiment. The experiment used digital image correlation (DIC) to measure the three-dimensional surface geometry and displacement field of the cornea at multiple pressures. A finite element model of a bovine cornea was developed using the DIC measured surface geometry of the undeformed specimen. The model was applied to determine five parameters of an anisotropic hyperelastic model that minimized the error between the measured and computed surface displacement field and to investigate the sensitivity of the measured bovine inflation response to variations in the anisotropic properties of the cornea. The results of the parameter optimization revealed that the collagen structure of bovine cornea exhibited a high degree of anisotropy in the limbus region, which agreed with recent histological findings, and a transversely isotropic central region. The parameter study showed that the bovine corneal response to the inflation experiment was sensitive to the shear modulus of the matrix at pressures below the intraocular pressure, the properties of the collagen lamella at higher pressures, and the degree of anisotropy in the limbus region. It was not sensitive to a weak collagen anisotropy in the central region.

Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice.

BackgroundKeratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation.Methodology/Principal FindingsHuman mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice.Conclusions/SignificanceUmbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea

In this study we present combined third-harmonic generation (THG) and second-harmonic generation (SHG) microscopy images of intact human corneas, and we analyze experimentally and theoretically the origin of the THG signal. Multiharmonic microscopy provides detailed images of the cornea microstructure over its entire thickness. A component of the THG signal originates from cellular structures and another one originates from anisotropy changes between successive collagen lamellae in the stroma. This anisotropy-related signal can be specifically detected using circular incident polarization, and provide contrasted images of the stacking and tissue-scale heterogeneity of stromal lamellae. Forward-radiated THG and SHG signals are generally anticorrelated, indicating that maximum THG is obtained from lamellar interfaces whereas maximum SHG is obtained from within lamellae. Polarization-resolved THG imaging reflects the a ernate anisotropy directions of the lamellae. We present a model for THG imaging of layered anisotropic samples and numerical calculations that account for our observations.

Multimodal Nonlinear Imaging of the Human Cornea

To evaluate the potential of third-harmonic generation (THG) microscopy combined with second-harmonic generation (SHG) and two-photon excited fluorescence (2PEF) microscopies for visualizing the microstructure of the human cornea and trabecular meshwork based on their intrinsic nonlinear properties. Fresh human corneal buttons and corneoscleral discs from an eye bank were observed under a multiphoton microscope incorporating a titanium-sapphire laser and an optical parametric oscillator for the excitation, and equipped with detection channels in the forward and backward directions. Original contrast mechanisms of THG signals in cornea with physiological relevance were elucidated. THG microscopy with circular incident polarization detected microscopic anisotropy and revealed the stacking and distribution of stromal collagen lamellae. THG imaging with linear incident polarization also revealed cellular and anchoring structures with micrometer resolution. In edematous tissue, a strong THG signal around cells indicated the local presence of water. Additionally, SHG signals reflected the distribution of fibrillar collagen, and 2PEF imaging revealed the elastic component of the trabecular meshwork and the fluorescence of metabolically active cells. The combined imaging modalities of THG, SHG, and 2PEF provide key information about the physiological state and microstructure of the anterior segment over its entire thickness with remarkable contrast and specificity. This imaging method should prove particularly useful for assessing glaucoma and corneal physiopathologies.

Microstructure Characteristics of the Cornea in Birds and Mammals

In this study, the microstructure of the cornea was compared among chickens (Gallus gallus), jungle crows (Corvus macrorhynchos), rats (Rattus norvegicus) and rabbits (Oryctolagus cuniculus). The density of keratocytes in the mammals was over 3 times that in the birds. The size of the keratocytes in the birds and rat were significantly lower than those in the rabbit. Using scanning and transmission electron microscopy, the bundles of collagen fibers in the birds were found to be well arranged, while those in the mammals were arranged randomly. The collagen lamellae of the birds were significantly thicker than those of the mammals, and the numbers of collagen lamellae in the birds were significantly smaller than in the mammals. The center-to-center distances between the collagen fibrils of the chicken and rabbit were significantly larger than those of the crow and rat. The densities of collagen fibrils in the chicken and rabbit were significantly less than those of the crow and rat.