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Epigenetic changes regulate developmental processes such as cell differentiation, X-chromosome inactivation and genomic imprinting. They also play an important role in modulating gene expression up or down in response to external (e.g., environmental) influences. Epigenetic processes in the placenta are quite distinct from other tissues, reflecting its unusual development and role as the interface between the mother and fetus. Investigation of the placental epigenome can add insight into (i) the unique aspects of placental development, including its relationship to metastatic processes in cancer; (ii) pathogenesis related to placental mediated pregnancy disorders; (iii) how in utero exposures, such as maternal nutrition, stress, drug use, and metabolic disease, can affect fetal health and long-term developmental outcomes; and (iv) possible utility in prenatal diagnosis.

The prevalence of the rotator cuff (RC) tears is ~ 21% in the general population, with higher incidences in individuals over 50. Irrespective of surgical repair techniques employed, re-tear rates are alarmingly high, indicating the need for improvement to the current treatment methods. A method that has recently increased in popularity is the administration of platelet-rich-plasma (PRP), as it has been proposed to significantly encourage and improve healing in a plethora of musculoskeletal tissues, although experimental conditions and results are often variable. This review aims to critically evaluate current literature concerning the use of PRP, specifically for the treatment of RC tears. There are ongoing conflicts debating the effectiveness of PRP to treat RC tears; with literature both in favour and against its use either having profound methodological weaknesses and/or limited applicability to most individuals with RC tears. There are numerous factors that may influence effectiveness, including the subgroup of patients studied and the timing and method of PRP delivery. Thus, in order to ascertain the clinical effectiveness of PRP for RC tears, the preparation protocol and composition of PRP must be standardised, so an accurate assessment and comparisons can be undertaken. Prior to clinical realisation, there is a requirement for a defined, standardised, quality-controlled protocol/procedure considering composition/formulation (of PRP); injury severity, dosage, frequency, timings, controls used, patient group, and rehabilitation programmes. Nevertheless, it is concluded that the initial step to aid the progression of PRP to treat RC tears is to standardise its preparation and delivery.

There is a current need for a therapy that can alleviate the social and economic burden that presents itself with debilitating and recurring musculoskeletal soft tissue injuries and disorders. Currently, several therapies are emerging and undergoing trials in animal models; these focus on the manipulation and administration of several growth factors implicated with healing. However, limitations include in vivo instability, reliance on biocompatible and robust carriers and restricted application procedures (local and direct). The aim of this paper is therefore to critically review the current literature surrounding the use of BPC 157, as a feasible therapy for healing and functional restoration of soft tissue damage, with a focus on tendon, ligament and skeletal muscle healing. Currently, all studies investigating BPC 157 have demonstrated consistently positive and prompt healing effects for various injury types, both traumatic and systemic and for a plethora of soft tissues. However, to date, the majority of studies have been performed on small rodent models and the efficacy of BPC 157 is yet to be confirmed in humans. Further, over the past two decades, only a handful of research groups have performed in-depth studies regarding this peptide. Despite this, it is apparent that BPC 157 has huge potential and following further development has promise as a therapy to conservatively treat or aid recovery in hypovascular and hypocellular soft tissues such as tendon and ligaments. Moreover, skeletal muscle injury models have suggested a beneficial effect not only for disturbances that occur as a result of direct trauma but also for systemic insults including hyperkalamia and hypermagnesia. Promisingly, there are few studies reporting any adverse reactions to the administration of BPC 157, although there is still a need to understand the precise healing mechanisms for this therapy to achieve clinical realisation.

Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing.

Rotator cuff tears can cause pain and weakness in the shoulder and affects a wide range of individuals. Conventional repair consists of operative surgical techniques or conservative treatments including physiotherapy; however, there is still a big issue in the increased risk of re-tearing of the tendons. Regenerative therapies are looking for new and novel ways of improving the healing of tendon injuries. Platelet-rich plasma (PRP) uses the patient’s own blood in a concentrated form, to supply the repair site with various growth factors that promote the healing response; however, research shows conflicting evidence about the efficacy and effectiveness of PRP. Stem cell research is slowly increasing use of human patients in research trials and is showing significant results in improving and reducing the re-tear rate. Tissue engineering encapsulates a range of techniques such as scaffolds and patches to augment repair of the rotator cuff tendon, and research has shown that such techniques can provide protection and assistance to the repairing tendon to improve results. This review aims to critically compare and evaluate recent research and provide possible future directions.

Background Placental inflammation, often presenting as acute chorioamnionitis (aCA), is commonly associated with preterm birth. Preterm birth can have both immediate and long-term adverse effects on the health of the baby. Developing biomarkers of inflammation in the placenta can help to understand its effects and potentially lead to new approaches for rapid prenatal diagnosis of aCA. We aimed to characterize epigenetic variation associated with aCA in placenta (chorionic villi) and fetal membranes (chorion and amnion) to better understand how aCA may impact processes that lead to preterm birth. This study lays the groundwork for development of novel biomarkers for aCA. Methods Samples from 44 preterm placentas (chorionic villi) as well as matched chorion and amnion for 16 of these cases were collected for this study. These samples were profiled using the Illumina Infinium HumanMethylation850 BeadChip to measure DNA methylation (DNAm) at 866,895 CpGs across the genome. An additional 78 placental samples were utilized to independently validate the array findings by pyrosequencing. Results Using a false discovery rate of 0.05, 66 differentially methylated (DM) CpG sites were identified between aCA cases and non-aCA cases in chorionic villi. For the majority of these 66 DM CpGs, the DNAm profile of the aCA cases as compared to the non-aCA cases trended in the direction of the blood cell DNAm. Interestingly, neutrophil-specific DNAm signatures, but not those associated with other immune cell types, were capable of separating aCA cases from the non-aCA cases. Conclusions Our results suggest that aCA-associated placentas showed altered DNAm signatures that were not observed in the absence of aCA. This DNAm profile is consistent with the activation of the innate immune response in the placenta and/or reflect increase in neutrophils as a response to inflammation. Electronic supplementary material The online version of this article (10.1186/s13072-018-0234-9) contains supplementary material, which is available to authorized users.

The placenta is a multifunctional organ that regulates key aspects of pregnancy maintenance and fetal development. As the placenta is in direct contact with maternal blood, cellular products (DNA, RNA, proteins, etc.) from the placenta can enter maternal circulation by a variety of ways. The application of serum proteins and circulating placental derived DNA has been well demonstrated for the diagnosis of aneuploidy, and there is great interest in exploring the use of placental biomarkers for the prediction of a range of fetal health parameters. In this review, we discuss how placental biomarkers might be used for the diagnosis and early detection of preeclampsia, fetal growth restriction and inflammation associated with preterm birth. We emphasize how increased understanding of the underlying placental biology can aid in the interpretation of such approaches and development of new biomarkers that can help predict the onset of pregnancy and neonatal health concerns before they manifest.

Purpose This review aimed to evaluate the efficacy of intra-articular injections of bone marrow derived mesenchymal stem cells (BM-MSCs) for the treatment of knee osteoarthritis (KOA). Methods This narrative review evaluates recent English language clinical data and published research articles between 2014 and 2019. Key word search strings of (((“bone marrow-derived mesenchymal stem cell” OR “bone marrow mesenchymal stromal cell” OR “bone marrow stromal cell”)) AND (“osteoarthritis” OR “knee osteoarthritis”)) AND (“human” OR “clinical”))) AND “intra-articular injection” were used to identify relevant articles using PMC, Cochrane Library, Web Of Science and Scopus databases. Results Pre-clinical studies have demonstrated successful, safe and encouraging results for articular cartilage repair and regeneration. This is concluded to be due to the multilineage differential potential, immunosuppressive and self-renewal capabilities of BM-MSCs, which have shown to augment pain and improve functional outcomes. Subsequently, clinical applications of intra-articular injections of BM-MSCs are steadily increasing, with most studies demonstrating a decrease in poor cartilage index, improvements in pain, function and Quality of Life (QoL); with moderate-to-high level evidence regarding safety for therapeutic administration. However, low confidence in clinical efficacy remains due to a plethora of heterogenous methodologies utilised, resulting in challenging study comparisons. A moderate number of cells (40 × 106) were identified as most likely to achieve optimal responses in individuals with grade ≥ 2 KOA. Likewise, significant improvements were reported when using lower (24 × 106) and higher (100 × 106) cell numbers, although adverse effects including persistent pain and swelling were a consequence. Conclusion Overall, the benefits of intra-articular injections of BM-MSCs were deemed to outweigh the adverse effects; thus, this treatment be considered as a future therapy strategy. To realise this, long-term large-scale randomised clinical trials are required to enable improved interpretations, to determine the validity of efficacy in future studies. Level of evidence IV.

Rotator cuff injuries (RCIs) present a major health problem due to high incidences of degenerative tears greater than 3 cm and prevalence of re-tears following surgical procedures. Since healing and functional restoration relies upon bone ingrowth into the tendon, it is hypothesised that sustained delivery of osteoinductive factors including bone morphogenetic proteins (BMPs), specifically BMP2–7, may significantly improve RCI tendon-bone healing. Here, growth factor candidates and delivery mechanisms are reviewed, specifically for improved RCI healing through enhanced bone ingrowth. In addition to BMPs, other potentially osteogenic factors including platelet-derived growth factors (PDGF), fibroblast growth factor (FGF), transforming growth beta isoforms (TGF-β1 and TGF-3) and parathyroid hormone (PTH) are evaluated since they can induce bone formation at the healing tendon attachment site. Several challenges must be addressed prior to clinical translation. The majority of published studies utilise in vivo animal models. In general, BMP-7 demonstrates a stronger stimulating effect when compared to BMP-2; the reported effectiveness of BMP-2 is often conflicting. Alternative factors, including PDGF and PTH, also demonstrate potential for assisting bone growth in enthesis healing. The use of sustained and biomimetic delivery systems appears to have the greatest positive effects. Some studies have demonstrated a dose-dependent effect, in conjunction with varying age, indicating that stratified therapies could be a viable solution for RCI healing. To adequately resolve potential treatments for RCI, further expanded and correlated animal trials must be undertaken, and indicative human trials are required with consideration of surgical and patient-specific influences.

The human pan-tissue epigenetic clock is widely used for estimating age across the entire lifespan, but it does not lend itself well to estimating gestational age (GA) based on placental DNAm methylation (DNAm) data. We replicate previous findings demonstrating a strong correlation between GA and genome-wide DNAm changes. Using substantially more DNAm arrays (n=1,102 in the training set) than a previous study, we present three new placental epigenetic clocks: 1) a robust placental clock (RPC) which is unaffected by common pregnancy complications (e.g., gestational diabetes, preeclampsia), and 2) a control placental clock (CPC) constructed using placental samples from pregnancies without known placental pathology, and 3) a refined RPC for uncomplicated term pregnancies. These placental clocks are highly accurate estimators of GA based on placental tissue; e.g., predicted GA based on RPC is highly correlated with actual GA (r>0.95 in test data, median error less than one week). We show that epigenetic clocks derived from cord blood or other tissues do not accurately estimate GA in placental samples. While fundamentally different from Horvath’s pan-tissue epigenetic clock, placental clocks closely track fetal age during development and may have interesting applications. publishedVersion

Osteoarthritis (OA) is a degenerative disease involving joint damage, an inadequate healing response and progressive deterioration of the joint architecture that commonly affects the knee and/or hip joints. It is a major world public health problem and is predicted to increase rapidly with an ageing population and escalating rate of obesity. Autologous blood-derived products possess much promise in the repair and regeneration of tissue and have important roles in inflammation, angiogenesis, cell migration and metabolism in pathological conditions, including OA. Utilising platelet-rich plasma (PRP) to treat tendon, ligament and skeletal muscle has shown variable results across many studies with the current evidence base for the efficacy of PRP in treating sports injuries remaining inconclusive. More uniformly positive results have been observed by various studies for PRP in OA knee in comparison to hyaluronic acid, other intra-articular injections and placebo than in other musculoskeletal tissue. However, methodological concerns as well as satisfactory PRP product classification prevent the true characterisation of this treatment. Thus, further research is required to investigate how leukocyte inclusion, activation and platelet concentration affect therapeutic efficacy. Furthermore, the optimisation of timing, dosage, volume, frequency and rehabilitation strategies need to be ascertained. For knee OA management, these concerns must be addressed before this promising treatment can be widely implemented.

Osteoarthritis (OA) is a common cause of chronic pain and disability. Regenerative therapies using mesenchymal stem cells (MSCs) provide an option for OA treatment as it could potentially regenerate the damaged cartilage. Bone marrow, adipose tissue and synovium are common MSC sources. The aim is to compare the therapeutic effect of MSCs from bone marrow, adipose tissue and synovium; combining its differentiation potential and accessibility, to decide the optimal source of MSCs for the treatment of knee OA. A comparison of preclinical and clinical studies using MSCs has been made with regard to treatment outcomes, isolation procedure and differentiation potential. All types of MSCs are effective at improving the clinical and structural condition of OA patients, but the longevity of the treatment, i.e. an effect that is maintained for at least 2 years, cannot be guaranteed. This review highlighted great variations in selection criteria and culture expansion conditions of MSCs between the literature and clinical trials. It also emphasised a substantial diversity and lack of consistency in the assessment mythology of clinical outcome after completion of MSC therapies procedures. A more cohesive methodology is required to evaluate the outcome of MSC treatments using quantitative and standardised frameworks in order to be able to directly compare results. Larger population of patients are recommended to assess the quality of MSC when designing studies and clinical trials to reaffirm the efficacy of MSC treatment prior to and within the clinical trials and follow up studies.

During pregnancy, the placenta releases a variety of nucleic acids (including deoxyribonucleic acid, messenger ribonucleic acid, or microribonucleic acids) either as a result of cell turnover or as an active messaging system between the placenta and cells in the maternal body. The profile of released nucleic acids changes with the gestational age and has been associated with maternal and fetal parameters. It also can directly reflect pathological changes in the placenta. Nucleic acids may therefore provide a rich source of novel biomarkers for the prediction of pregnancy complications. However, their utility in the clinical setting depends, first, on overcoming some technical considerations in their quantification, and, second, on developing a better understanding of the factors that influence their function and abundance.

Psychological challenges, including traumatic events, have been hypothesized to increase the age-related pace of biological aging. Here we test the hypothesis that psychological challenges can affect the pace of telomere attrition, a marker of cellular aging, using data from an ongoing longitudinal-cohort study of Kaqchikel Mayan women living in a population with a high frequency of child mortality, a traumatic life event. Specifically, we evaluate the associations between child mortality, maternal telomere length and the mothers’ hypothalamic-pituitary-adrenal axis (HPAA), or stress axis, activity. Child mortality data were collected in 2000 and 2013. HPAA activity was assessed by quantifying cortisol levels in first morning urinary specimens collected every other day for seven weeks in 2013. Telomere length (TL) was quantified using qPCR in 55 women from buccal specimens collected in 2013. Results: Shorter TL with increasing age was only observed in women who experienced child mortality (p = 0.015). Women with higher average basal cortisol (p = 0.007) and greater within-individual variation (standard deviation) in basal cortisol (p = 0.053) presented shorter TL. Non-parametric bootstrapping to estimate mediation effects suggests that HPAA activity mediates the effect of child mortality on TL. Our results are, thus, consistent with the hypothesis that traumatic events can influence cellular aging and that HPAA activity may play a mediatory role. Future large-scale longitudinal studies are necessary to confirm our results and further explore the role of the HPAA in cellular aging, as well as to advance our understanding of the underlying mechanisms involved.

The worldwide limited availability of suitable corneal donor tissue has led to the development of alternatives, including keratoprostheses (Kpros) and tissue engineered (TE) constructs. Despite advances in bioscaffold design, there is yet to be a corneal equivalent that effectively mimics both the native tissue ultrastructure and biomechanical properties. Human decellularized corneas (DCs) could offer a safe, sustainable source of corneal tissue, increasing the donor pool and potentially reducing the risk of immune rejection after corneal graft surgery. Appropriate, human-specific, decellularization techniques and high-resolution, non-destructive analysis systems are required to ensure reproducible outputs can be achieved. If robust treatment and characterization processes can be developed, DCs could offer a supplement to the donor corneal pool, alongside superior cell culture systems for pharmacology, toxicology and drug discovery studies.

Author(s): Albrecht, Christiane; Baker, Julie C; Blundell, Cassidy; Chavez, Shawn L; Carbone, Lucia; Chamley, Larry; Hannibal, Roberta L; Illsley, Nick; Kurre, Peter; Laurent, Louise C; McKenzie, Charles; Morales-Prieto, Diana; Pantham, Priyadarshini; Paquette, Alison; Powell, Katie; Price, Nathan; Rao, Balaji M; Sadovsky, Yoel; Salomon, Carlos; Tuteja, Geetu; Wilson, Samantha; O'Tierney-Ginn, PF | Abstract: Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops covered innovative technologies applied to new and traditional areas of placental research: 1) genomic communication; 2) bioinformatics; 3) trophoblast biology and pathology; 4) placental transport systems.

Control and maintenance of the keratocyte phenotype is vital to developing in vitro tissue engineered strategies for corneal repair. In this study the influence of topographical and chemical cues on the mechanical, phenotypical and genotypical behaviour of adult human derived corneal stromal (AHDCS) cells in three dimensional (3D) multi-layered organised constructs is examined. Topographical cues are provided via multiple aligned electrospun nanofiber meshes, which are arranged orthogonally throughout the constructs and are capable of aligning individual cells and permitting cell migration between the layers. The influence of chemical cues is examined using different supplements in culture media. A non-destructive indentation technique and optical coherence tomography are used to determine the matrix elasiticity (elastic modulus) and dimensional changes, respectively. These measurements were indicative of changes in cell phenotype from contractile fibroblasts to quiescent keratocytes over the duration of the experiment and corroborated by qPCR. Constructs containing nanofibers have a higher initial modulus, reduced contraction and organised cell orientation compared to those without nanofibers. Cell-seeded constructs cultured in serum-containing media increased in modulus throughout the culture period and underwent significantly more contraction than constructs cultured in serum-free and insulin-containing media. This implies that the growth factors present in serum promote a fibroblast-like phenotype; qPCR data further validates these observations. These results indicate that the synergistic effect of nanofibers and serum-free media plus insulin supplementation provide the most suitable topographical and chemical environment for reverting corneal fibroblasts to a keratocyte phenotype in a 3D construct.

Background Early detection of pregnancies at risk of complications, such as intrauterine growth restriction (IUGR) and preeclampsia (PE), is critical for improved monitoring and preventative treatment to optimize health outcomes. We predict that levels of placental-derived proteins circulating in maternal blood reflect placental gene expression, which is associated with placental DNA methylation (DNAm) profiles. As such, placental DNAm profiling may be useful to distinguish pregnancies at risk of developing complications and correlation between DNAm and protein levels in maternal blood may give further evidence for a protein’s use as a biomarker. However, few studies investigate all clinical parameters that may influence DNAm and/or protein expression, which can significantly affect the relationship between these measures. Results Candidate genes were chosen based on i) reported alterations of protein levels in maternal blood and ii) observed changes in placental DNAm (∆β > 0.05 and False Discovery Rate (FDR)

PURPOSE. Corneal decellularization has emerged as a promising alternative to traditional tissue-engineering strategies for the creation of corneal replacements for transplantation. However, decellularization methods can lead to swelling of the cornea, limiting its potential use as a scaffold. In this study, we propose the use of a complex polysaccharide, dextran, to reduce this swelling and maintain the native dimensions and architecture of the cornea. METHODS. Porcine corneal buttons were treated with Triton X-100, SDS and nucleases under constant rotation followed by a washing step. To prevent corneal swelling, the decellularization solution was supplemented with dextran. This solution was added to one group throughout the decellularization process and to a second group during the washing cycle. The resulting acellular scaffolds were systematically evaluated by histological and biochemical analyses, in addition, the ultrastructure of the cornea was examined by transmission electron microscopy (TEM). RESULTS. Results demonstrated that the combination of detergents and nucleases effectively removed the majority of cellular material from the cornea. Furthermore, the addition of dextran prevented significant swelling when used throughout the protocol or only during the washing process. After soaking in glycerol a degree of transparency was returned to all decellularized corneas suggesting maintenance of the extracellular matrix. However, TEM analysis confirmed that dextran must be present throughout the decellularization process to preserve the native ultrastructure of the cornea. CONCLUSIONS. The findings of this study indicate that the addition of dextran to the decellularization process not only prevents significant corneal swelling but also enhances the maintenance of the native ultrastructure of the cornea.

The current worldwide cornea shortages have led to the development of feasible, long-term substitutes to cadaveric donor tissue. Amniotic membrane is a clinically successful material used to reconstruct damaged corneal surfaces. However, its reliance upon the host tissue, availability, donor variation, infection risks and the fact that it is currently unsuitable for complete corneal regeneration have warranted the need for alternative solutions. Alternatives in current clinical or pre-clinical development include keratoprostheses, tissue engineered constructs, xenografts and the use of acellular matrices. With respect to corneal regeneration, there are many challenges, not least that the corneal structure is unique and difficult to replicate. When manufacturing corneal tissues, the choice of material is vital as the list of requirements is extensive. They must be biocompatible, (preferably) optically transparent, flexible, and strong, as to withstand manipulation in culture, potential suturing, irrigation and handling during surgery. Manufacturing process need to be simple and consistent, preferably at high speed and low cost. Two fundamental objectives of corneal regeneration are the maintenance of healthy cell phenotypes and the replication of the native tissue architecture. If both factors are not satisfied, the result is often regenerated tissue mimicking that of scarred native tissue.

In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-β1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial-stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-β1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-β1 media supplementation. It was then revealed that epithelial-stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal-epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital.

Corneal structure is highly organized and unified in architecture with structural and functional integration which mediates transparency and vision. Disease and injury are the second most common cause of blindness affecting over 10 million people worldwide. Ninety percent of blindness is permanent due to scarring and vascularization. Scarring caused via fibrotic cellular responses, heals the tissue, but fails to restore transparency. Controlling keratocyte activation and differentiation are key for the inhibition and prevention of fibrosis. Ophthalmic surgery techniques are continually developing to preserve and restore vision but corneal regression and scarring are often detrimental side effects and long term continuous follow up studies are lacking or discouraging. Appropriate corneal models may lead to a reduced need for corneal transplantation as presently there are insufficient numbers or suitable tissue to meet demand. Synthetic optical materials are under development for keratoprothesis although clinical use is limited due to implantation complications and high rejection rates. Tissue engineered corneas offer an alternative which more closely mimic the morphological, physiological and biomechanical properties of native corneas. However, replication of the native collagen fiber organization and retaining the phenotype of stromal cells which prevent scar-like tissue formation remains a challenge. Careful manipulation of culture environments are under investigation to determine a suitable environment that simulates native ECM organization and stimulates keratocyte migration and generation.

The effect of different collagen and cell concentrations on the mechanical and remodeling behaviors of corneal stroma wound healing models consisting of collagen hydrogels seeded with human corneal fibroblasts during a 25 day culture period were examined. Human corneal fibroblasts were seeded at 1 × 10(5), 3 × 10(5) or 5 × 10(5) cells per hydrogel, and collagen concentrations of 2.5 mg/ml, 3.5 mg/ml or 4.5 mg/ml were examined. Two non-destructive techniques, spherical indentation and optical coherence tomography, were used to measure the elastic modulus and dimensional changes respectively at several time-points over the culture period. The elastic modulus of the hydrogels increased continuously over 25 days. Hydrogels with higher initial cell seeding densities and lower initial collagen concentrations were found to increase in elastic modulus faster and possessed a higher elastic modulus by the end of the culture period when compared to the other hydrogels. A mathematical equation was applied to accurately fit the change in elastic modulus over time. This study demonstrates a robust in vitro technique able to monitor the effect of different parameters on the cell-matrix mechanical relationship in a corneal stroma model during prolonged culture periods and enhances our understanding on corneal wound healing processes.