Differentiated activities of decorin and biglycan in the progression of post-traumatic osteoarthritis.

OBJECTIVE: To delineate the activities of decorin and biglycan in the progression of post-traumatic osteoarthritis (PTOA). DESIGN: Three-month-old inducible biglycan (BgniKO) and decorin/biglycan compound (Dcn/BgniKO) knockout mice were subjected to the destabilization of the medial meniscus (DMM) surgery to induce PTOA. The OA phenotype was evaluated by assessing joint structure and sulfated glycosaminoglycan (sGAG) staining via histology, surface collagen fibril nanostructure and calcium content via scanning electron microscopy, tissue modulus via atomic force microscopy-nanoindentation, as well as subchondral bone structure and meniscus ossification via micro-computed tomography. Outcomes were compared with previous findings in the inducible decorin (DcniKO) knockout mice. RESULTS: In the DMM model, BgniKO mice developed similar degree of OA as the control (0.44 [-0.18 1.05] difference in modified Mankin score), different from the more severe OA phenotype observed in DcniKO mice (1.38 [0.91 1.85] difference). Dcn/BgniKO mice exhibited similar histological OA phenotype as DcniKO mice (1.51 [0.97 2.04] difference vs control), including aggravated loss of sGAGs, salient surface fibrillation and formation of osteophyte. Meanwhile, Dcn/BgniKO mice showed further cartilage thinning than DcniKO mice, resulting in the exposure of underlying calcified tissues and aberrantly high surface modulus. BgniKO and Dcn/BgniKO mice developed altered subchondral trabecular bone structure in both Sham and DMM groups, while DcniKO and control mice did not. CONCLUSION: In PTOA, decorin plays a more crucial role than biglycan in regulating cartilage degeneration, while biglycan is more important in regulating subchondral bone structure. The two have distinct activities and modest synergy in the pathogenesis of PTOA.

Mindfulness-based stress reduction adapted to pregnant women with psychosocial vulnerabilities-a protocol for a randomized feasibility study in a Danish hospital-based outpatient setting.

BACKGROUND: This protocol is for a feasibility study of a mindfulness-based stress reduction (MBSR) program adapted for pregnant women with psychosocial vulnerabilities. The rationale for the study is the need for a wider array of evidence-based options to address prenatal mental health care needs in pregnant women. MBSR is a promising mental health intervention but has not yet been adapted for pregnant women with the aim of addressing prenatal mental health. The purpose is thus to evaluate the feasibility, acceptability, and clinical outcomes of an adapted MBSR program, prenatal MBSR, compared to usual care to inform a randomized controlled trial. METHODS/DESIGN: Pregnant women (n = 60) referred to an outpatient clinic at Copenhagen University Hospital, Amager and Hvidovre, Denmark, will be recruited for the study. The design is a single-center feasibility trial, with prenatal MBSR, as an add-on to usual care. The primary outcome is to assess the feasibility of a full-scale randomized controlled trial. The secondary feasibility outcome includes possible effects of the adapted MBSR program estimated by self-report questionnaires measuring stress, anxiety, depression, well-being, decentering, reflective functioning, mindfulness, and compassion. Participants will be randomized in a 1:1 ratio to prenatal MBSR or usual care. DISCUSSION: The study is part of the Good Start to Family Life study anchored at Copenhagen University Hospital, Amager and Hvidovre, Denmark. Teaching the skills of mindfulness meditation to a psychosocially vulnerable group of pregnant women could prove a viable and non-pharmacological approach to reduce stress, improve mental health, and provide support in the transition to parenthood. The outcomes of the feasibility study will inform the design of a fully powered randomized controlled trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04571190 . Registered on September 30, 2020.

Training in Bariatric Surgery: a National Survey of German Bariatric Surgeons.

INTRODUCTION: Skill in bariatric surgery has been associated with postoperative outcome. Appropriate surgical training is of paramount importance. In order to continuously improve training strategies, it is necessary to assess current practices. AIM: To determine how German bariatric surgeons have been trained and to assess current training strategies. METHODS: Between February 2017 and March 2017, an online census of surgeons registered as members of the German Society for Bariatric and Metabolic Surgery was conducted. A total of three reminders were sent out. Data were analyzed using descriptive statistics. Data was reported as median (interquartile range); percentages were adjusted for completed answers only. RESULTS: A response rate of 51% (n = 214) was achieved. Surgeons reported a median of 14.5 (8-20) years of surgical experience after initial training, with a specific bariatric experience of 7 (4-13) years. The total cumulative bariatric case volume was 240 (80-500) cases, with an annual case volume of 50 (25-80). The most commonly applied approaches to bariatric skills acquisition were "learning by doing" (71%), "course participation" (70%) and "observerships" (70%). Fellowships and the use of operating videos were less frequently applied strategies (19%/ 47%). Interestingly, observerships (94%) and course participation (89%) were rated as very important/important, whereas "learning by doing" (62%), watching operation videos (59%), and fellowships (48%) were less frequently perceived as important/very important training strategies. CONCLUSIONS: The majority of surgeons performing bariatric cases were senior surgeons with more than 10 years of post-training experience; nevertheless, the survey revealed a lack of structured approaches to bariatric specialization training.

A nationwide survey on bariatric surgery in Germany--results 2005-2007.

BACKGROUND: Most studies on bariatric surgery outcomes are performed as clinical trials or reflect the clinical experience in single centers. The status of bariatric surgery in Germany has been examined since January 1st, 2005 with the cooperation of clinics and hospitals at the Institute of Quality Assurance in Surgery at the Otto-von-Guericke University of Magdeburg (Germany). METHODS: In this prospective multicenter observational study, the data obtained for all primary bariatric procedures, including all repeated operations, performed on consecutive patients with morbid obesity at participating hospitals from 2005 to 2007 were prospectively collected using an Internet online data registry. In particular, perioperative characteristics, such as the spectrum of diagnostic measurements, type of surgical procedures, and short- and long-term outcomes, were investigated. RESULTS: During the study period, 629 surgical procedures were performed at 21 hospitals in 2005, 828 procedures at 32 hospitals in 2006, and 1,666 procedures at 35 hospitals in 2007. In 2005 and 2006, gastric banding was the most frequently performed operation, followed by the Roux-en-Y gastric bypass (RYGBP). In 2007, a RYGBP was carried out in 42.1% of all bariatric procedures. Among all patients, 74.4% were female. The mean body mass index (BMI) was 48.5 kg/m(2) in 2005, 48.4 kg/m(2) in 2006, and 48.0 kg/m(2) in 2007. Follow-up data after 12 months were available for 63.8% of the patients in 2005 and 2006; these data showed greater reduction of BMI after malabsorptive rather than restrictive bariatric procedures. The mortality was 0.1% (30 days) and 0.16% (overall). CONCLUSION: As indicated by the worldwide trend, there is an ongoing change from restrictive bariatric procedures to malabsorptive procedures and sleeve gastrectomy. Although the BMIs of German patients undergoing bariatric surgery appear to be substantially higher than those of patients from most other countries, there were no differences in intraoperative and short-term complications or in overall outcomes during follow-up when compared with published studies.

Results of sleeve gastrectomy-data from a nationwide survey on bariatric surgery in Germany.

BACKGROUND: Beginning January 1, 2005, the status and outcomes of bariatric surgery were examined in Germany. Data are registered in cooperation with the An-Institute of quality assurance in surgery at the Otto-von-Guericke-University Magdeburg. The objective of this study was to examine the morbidity and mortality rates secondary to sleeve gastrectomy (SG) in Germany since 2006. METHODS: Data collection occurred prospectively in an online data bank. All primary bariatric procedures performed were recorded as were all re-operations in patients that had already undergone a primary operation. Specific data compiled on the sleeve gastrectomy procedure were evaluated with a focus on operative details and complication rates. RESULTS: The total study cohort contains 3,122 patients. From January 2006 to December 2007, 144 sleeve gastrectomy procedures were performed in the 17 hospitals participating in the study. The mean body mass index (BMI) of all patients was 48.8 kg/m(2). The BMI of patients undergoing SG was 54.5 kg/m(2). In total, 73.8% of the patients were female and 26.2% of the patients were male. There were no significant differences between patients undergoing SG. The general complication rate after SG was 14.1%, and the surgical complication rate was 9.4%. The postoperative mortality rate was 1.4%. CONCLUSIONS: The complication rate during the first 2 years after SG in Germany is similar to that published in the literature. In order to improve the quality of bariatric surgery, an evaluation of data from a German multicenter trial is necessary to evaluate the position of SG in the bariatric algorithm.

Evidence of thromboembolism prophylaxis in bariatric surgery-results of a quality assurance trial in bariatric surgery in Germany from 2005 to 2007 and review of the literature.

BACKGROUND: Since January 1st, 2005, the current situation for bariatric surgery has been examined by means of a voluntary quality assurance study in Germany with a multicenter design in which 38 hospitals and surgical departments participated. The data are registered in cooperation with the Institute of Quality Assurance in Surgery at the Otto-von-Guericke University of Magdeburg (Germany). METHODS: Data describing peri-interventional characteristics were prospectively documented in an internet online data registry. All primary bariatric procedures performed since January 1st, 2005, were registered. In addition, reoperations in patients who had previously undergone primary surgical intervention were included. As a representative excerpt from the overall prospective multicenter observational study on obesity surgery, data on the type, regimen, and time course of deep venous thrombosis (DVT) prophylaxis were documented. From the number and spectrum of complications, the incidences of clinically manifest DVT or pulmonary embolism (PE) were derived during the in-hospital course and follow-up in conjunction with the type of surgical procedure and body mass index (BMI). RESULTS: Overall, 3,122 bariatric procedures were performed at 38 German hospitals between January 2005 and December 2007. These procedures were subdivided into 2,869 primary operations and 253 revisions (sex ratio, male to female = 25.6:74.4%). The average BMI of all patients was 48.5 kg/m(2) in 2005, 48.4 kg/m(2) in 2006, and 48.0 kg/m(2) in 2007. In 2005 and 2006, gastric banding (GB) was the most commonly performed operation, followed by Roux-en-Y gastric bypass (RYGBP). In 2007, RYGBP was carried out in 42.1% of all bariatric procedures. Interestingly, the incidence of deep venous thrombosis (DVT) was only 0.06%, whereas PE occurred in 0.06% of patients only after hospital discharge. The DVT prophylaxis protocol used has been changed for the last 2 years: the majority of patients with a BMI above 50 kg/m(2) received low-molecular-weight heparin twice a day. CONCLUSION: In Germany, a trend from GB to sleeve gastrectomy (SG) and malabsorptive approach has been evaluated. This trend is associated with differences of the DVT prophylaxis regimen in the profile of bariatric surgical patients depending on BMI and the type of bariatric procedure. Despite the low incidence of DVT and pulmonary embolism (PE) detected, there is a lack of evidence on a reasonable regimen for sufficient DVT prophylaxis in bariatric surgery; instead, there are only recommendations from the guidelines and statements of a specific medical society. Therefore, prospective studies are necessary to determine the optimal DVT prophylaxis for bariatric surgical patients as well as obese patients undergoing surgery.

Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation.

The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts.

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.

Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons.

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.

Collagen type I and type V are present in the same fibril in the avian corneal stroma.

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.

Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology.

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.

The spatial organization of Descemet's membrane-associated type IV collagen in the avian cornea.

The organization of type IV collagen in the unconventional basement membrane of the corneal endothelium (Descemet's membrane) was investigated in developing chicken embryos using anti-collagen mAbs. Both immunofluorescence histochemistry and immunoelectron microscopy were performed. In mature embryos (greater than 15 d of development), the type IV collagen of Descemet's membrane was present as an array of discrete aggregates of amorphous material at the interface between Descemet's membrane and the posterior corneal stroma. Immunoreactivity for type IV collagen was also observed in the posterior corneal stroma as irregular plaques of material with a morphology similar to that of the Descemet's membrane-associated aggregates. This arrangement of Descemet's membrane-associated type IV collagen developed from a subendothelial mat of type IV collagen-containing material. This mat, in which type IV collagen-specific immunoreactivity was always discontinuous, first appeared at the time a confluent endothelium was established, well before the onset of Descemet's membrane formation. Immunoelectron microscopy of mature corneas revealed that the characteristic nodal matrix of Descemet's membrane itself was unreactive for type IV collagen, but was penetrated at intervals by projections of type IV collagen-containing material. These projections frequently appeared to contact cell processes from the underlying corneal endothelium. This spatial arrangement of type IV collagen suggests that it serves to suture the corneal endothelium/Descemet's membrane to the dense interfacial matrix of the posterior stroma.

Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis.

Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b) the high content of type V collagen within the corneal collagen fibrils is one factor responsible for the small, uniform fibrillar diameter (25 nm) characteristic of this tissue, (c) the completely processed form of type V collagen found within tissues retains a large noncollagenous region, termed the NH2-terminal domain, at the amino end of its alpha 1 chain, and (d) the NH2-terminal domain may contain at least some of the information for the observed regulation of fibril diameters. In the present investigation we have employed polyclonal antibodies against the retained NH2-terminal domain of the alpha 1(V) chain for immunohistochemical studies of embryonic avian corneas and for immunoscreening a chicken cDNA library. When combined with cDNA sequencing and molecular rotary shadowing, these approaches provide information on the molecular structure of the retained NH2-terminal domain as well as how this domain might function in the regulation of fibrillar structure. In immunofluorescence and immunoelectron microscopy analyses, the antibodies against the NH2-terminal domain react with type V molecules present within mature heterotypic fibrils of the corneal stroma. Thus, epitopes within at least a portion of this domain are exposed on the fibril surface. This is in marked contrast to mAbs which we have previously characterized as being directed against epitopes located in the major triple helical domain of the type V molecule. The helical epitopes recognized by these antibodies are antigenically masked on type V molecules that have been assembled into fibrils. Sequencing of the isolated cDNA clones has provided the conceptual amino acid sequence of the entire amino end of the alpha 1(V) procollagen chain. The sequence shows the location of what appear to be potential propeptidase cleavage sites. One of these, if preferentially used during processing of the type V procollagen molecule, can provide an explanation for the retention of the NH2-terminal domain in the completely processed molecule. The sequencing data also suggest that the NH2-terminal domain consists of several regions, providing a structure which fits well with that of the completely processed type V molecule as visualized by rotary shadowing.

[Barriers to the German Society for General and Visceral Surgery (DGAV) accreditation "Center for bariatric and metabolic surgery"]. / Barrieren auf dem Weg zur DGAV-Zertifizierung "Zentrum für Adipositas- und metabolische Chirurgie".

INTRODUCTION: The certification and accreditation process of the German Society for General and Visceral Surgery (DGAV) aims to improve the quality of care and enhance transparency in accredited centers. To achieve these goals a high level of infrastructural and staffing requirements are set out by the DGAV. AIMS: The Surgical Working Group on Obesity Treatment and Metabolic Surgery (CAADIP) survey 2017 of the members of the German Bariatric Society aimed to identify the perceived and encountered barriers in the DGAV accreditation process. METHODS: Between February and March 2017 an online poll was conducted of all members of the CAADIP on topics pertaining to the accreditation process. RESULTS: A total of 214 (51%) CAADIP members participated in the poll, 47% reported that they worked at a non-certified center and 53% worked at a DGAV certified center. Of these, 68% reported employment in a unit with the lowest accreditation level, 21% in an intermediate level center, 11% reported employment in a unit with the highest accreditation level (Center of Excellence) and 86% of those currently working in non-accredited units stated that they aimed for future accreditation. Reasons stated for not having obtained accreditation were recent establishment of the new bariatric specialty (54%), lack of bariatric case numbers (71%), lack of human resources and infrastructure (28% and 13%, respectively). Of those surgeons in non-accredited centers 24% stated that the hospital management had no interest in a certification and 12% of the surgeons themselves felt that accreditation was unnecessary. CONCLUSION: The majority of CAADIP members strived for DGAV certification. The main barriers encountered and perceived were the specific time (reference years) and caseload requirements.

Altered wound healing in mice lacking a functional osteopontin gene (spp1).

Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)- containing glycoprotein encoded by the gene secreted phosphoprotein 1 (spp1). spp1 is expressed during embryogenesis, wound healing, and tumorigenesis; however, its in vivo functions are not well understood. Therefore, OPN null mutant mice were generated by targeted mutagenesis in embryonic stem cells. In OPN mutant mice, embryogenesis occurred normally, and mice were fertile. Since OPN shares receptors with vitronectin (VN), we tested for compensation by creating mice lacking both OPN and VN. The double mutants were also viable, suggesting that other RGD-containing ligands replace the embryonic loss of both proteins. We tested the healing of OPN mutants after skin incisions, where spp1 was upregulated as early as 6 h after wounding. Although the tensile properties of the wounds were unchanged, ultrastructural analysis showed a significantly decreased level of debridement, greater disorganization of matrix, and an alteration of collagen fibrillogenesis leading to small diameter collagen fibrils in the OPN mutant mice. These data indicate a role for OPN in tissue remodeling in vivo, and suggest physiological functions during matrix reorganization after injury.

Nuclear ferritin protects DNA from UV damage in corneal epithelial cells.

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3'-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

Corneal and scleral collagen fiber formation in vitro.

We have investigated the role of structural differences in collagen molecules and the effect of proteoglycan preparations on the control of collagen fibril formation. Collagen and proteoglycans were extracted, purified and characterized from two structurally and functionally different connective tissues, rabbit corneal stroma and sclera. Corneal collagen was found to form fibers 6- to 7-times more slowly than scleral type I collagen. Proteoglycans from both sources retard fibrillogenesis, with corneal proteoglycans having approximately 3-times the effect observed with scleral proteoglycans. The morphology of the fibers formed was normal in all cases. Therefore, the changes observed may reflect a true control mechanism related to the strict morphological arrangement associated with corneal transparency.

Development and roles of collagenous matrices in the embryonic avian cornea.

Corneal development requires the production, assembly and sometimes replacement of a number of collagenous matrices. The embryonic chick cornea is well-characterized and offers certain advantages for studying the assembly and roles of these matrices. We will first describe the matrices to be examined. These include the corneal stroma proper, first formed as the primary stroma and subsequently as the secondary (mature) stroma; Bowman's Membrane; Descemet's Membrane; and the hemidesmosome of the epithelial cell attachment complex. We will then describe the characteristics of the collagen types involved, including: the fibrillar collagens (types I, II and V), the fibril-associated collagens (types IX, XII and XIV), and the transmembrane collagen of the hemidesmosome (type XVII). Then, in each subsequent section we will examine in detail the structure, assembly and development of each collagenous matrix, and how each specific collagen and/or combination of collagens are thought to provide the matrices with their unique properties. The work and views presented here are largely from our own laboratories. Thus, this article is not meant to be a comprehensive review of the literature. For pertinent references by others, when possible, we will cite recent reviews.

Development of tendon structure and function: regulation of collagen fibrillogenesis.

In the tendon, the development of mature mechanical properties is dependent on the assembly of a tendon-specific extracellular matrix. This matrix is synthesized by the tendon fibroblasts and composed of collagen fibrils organized as fibers, as well as fibril-associated collagenous and non-collagenous proteins. All of these components are integrated, during development and growth, to form a functional tissue. During tendon development, collagen fibrillogenesis and matrix assembly progress through multiple steps where each step is regulated independently, culminating in a structurally and functionally mature tissue. Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and mature fibrils grow through a process of post-depositional fusion of the intermediates. Linear and lateral fibril growth occurs after the immature fibril intermediates are incorporated into fibers. The processes are regulated by interactions of extracellular macromolecules with the fibrils. Interactions with quantitatively minor fibrillar collagens, fibril-associated collagens and proteoglycans influence different steps in fibrillogenesis and the extracellular microdomains provide a mechanism for the tendon fibroblasts to regulate these extracellular interactions.