Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids.

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Rubidium-Containing Calcium Alginate Hydrogel for Antibacterial and Diabetic Skin Wound Healing Applications.

Rubidium (Rb) is an important microelement for the human body, which can kill and inhibit bacteria. Rubidium-containing polyhydrated ionogens have been applied to treat refractory wounds. In this work, for the first time, Rb was added into calcium alginate hydrogel (Rb-CA gel) and then made into a dressing by freeze-drying. The addition of Rb to CA maintained a well-ordered porous structure with only slightly reduced swelling and storage modulus and increased roughness and in vitro degradation rate. Experiments in vitro demonstrated that Rb-CA gels could not only inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa ubiquitous but were nontoxic and even conducive to human umbilical vein endothelial cells (HUVECs), fibroblasts, and keratinocytes. HUVECs exposed to Rb-CA gels exhibited enhanced migration and tubule formation ability, increased vascular endothelial growth factor secretion, and improved activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2)/heme-oxygenase-1 (HO-1) signaling pathway. Rb-CA gels also promoted the migration of fibroblasts and keratinocytes. In vivo, in a type II diabetic male Sprague-Dawley rat wound model, neovascularization, re-epithelialization, and collagen deposition were all improved greatly by the Rb-CA gel, with the upregulation of NRF2 and HO-1 protein secretion that inhibited the oxidative stress reaction in the wound sites. Moreover, the Rb-CA gel exhibited a strong anti-inflammatory effect on the wound. In summary, the application of Rb-CA gel dressings to diabetic wounds could produce various synergetic enhancements in terms of angiogenesis, re-epithelialization, and collagen deposition. Hence, Rb-CA gels can be used as a novel therapeutic strategy to promote the healing of diabetic wounds.

The Application of Biomaterials to Tissue Engineering Neural Retina and Retinal Pigment Epithelium.

The prevalence of degenerative retinal disease is ever increasing as life expectancy rises globally. The human retina fails to regenerate and the use of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) to engineer retinal tissue is of particular interest due to the limited availability of suitable allogeneic or autologous tissue. Retinal tissue and its development are well characterized, which have resulted in robust assays to assess the development of tissue-engineered retina. Retinal tissue can be generated in vitro from hESCs and hiPSCs without biomaterial scaffolds, but despite advancements, protocols remain slow, expensive, and fail to result in mature functional tissue. Several recent studies have demonstrated the potential of biomaterial scaffolds to enhance generation of hESC/hiPSC-derived retinal tissue, including synthetic polymers, silk, alginate, hyaluronic acid, and extracellular matrix molecules. This review outlines the advances that have been made toward tissue-engineered neural retina and retinal pigment epithelium (RPE) for clinical application in recent years, including the success of clinical trials involving transplantation of cells and tissue to promote retinal repair; and the evidence from in vitro and animal studies that biomaterials can enhance development and integration of retinal tissue.

Extracellular matrix component expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix.

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. STATEMENT OF SIGNIFICANCE: The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation.

3D culture of human pluripotent stem cells in RGD-alginate hydrogel improves retinal tissue development.

No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is, however, limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel), 0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker, MATH5. Furthermore, 0.5% RGD-alginate hydrogel encapsulation did not adversely affect the expression of other neural retina markers (PROX1, CRX, RCVRN, AP2α or VSX2) as determined by qRT-PCR, or the percentage of VSX2 positive cells as determined by flow cytometry. 1% RGD-alginate increased the percentage of EBs with OVs and/or RPE, but did not significantly influence any other measures of retinal differentiation. HA-based hydrogels had no significant effect on retinal tissue development. The results indicated that derivation of retinal tissue from hESCs/hiPSCs can be enhanced by culture in 0.5% RGD-alginate hydrogel. This RGD-alginate scaffold may be useful for derivation, transport and transplantation of neural retina and RPE, and may also enhance formation of other pigmented, neural or epithelial tissue. STATEMENT OF SIGNIFICANCE: The burden of retinal disease is ever growing with the increasing age of the world-wide population. Transplantation of retinal tissue derived from human pluripotent stem cells (PSCs) is considered a promising treatment. However, derivation of retinal tissue from PSCs using defined media is a lengthy process and often variable between different cell lines. This study indicated that alginate hydrogels enhanced retinal tissue development from PSCs, whereas hyaluronic acid-based hydrogels did not. This is the first study to show that 3D culture with a biomaterial scaffold can improve retinal tissue derivation from PSCs. These findings indicate potential for the clinical application of alginate hydrogels for the derivation and subsequent transplantation retinal tissue. This work may also have implications for the derivation of other pigmented, neural or epithelial tissue.

Lack of evidence to support present medial release methods in total knee arthroplasty.

PURPOSE: The aim of this review was to identify a reliable sequential medial release protocol for restoration of soft tissue balance in total knee arthroplasty of the varus osteoarthritic knee and to allow for improved intraoperative decision-making. METHOD: Current medial release sequences and applicability based upon pre-operative deformity have been reviewed. Furthermore, risks associated with over release, and the necessity of medial release, are discussed. RESULTS: The different medial release sequences are discussed in relation to pre-operative deformity, along with potential complications associated with medial release. It was found that release sequences may include the deep and superficial components of the medial collateral ligament, the posteromedial capsule, the posterior oblique ligament, the pes anserinus (pes A), and tendons of the semimembranosus and medial gastrocnemius muscle. The sequences described were found to vary substantially between studies, and very few studies had systematically quantified the effect of each release on balance. CONCLUSION: While medial release is the standard intraoperative mode of balancing, there is a lack of evidence to support current methods. The correct method for defining intraoperatively the sequence, extent and magnitude of releases required remains ill-defined. It could be argued that the classic extensive medial release may be unnecessary and may be associated with iatrogenic injury to the pes A and saphenous nerve, instability and abnormal knee kinematics. Minimal medial release may allow for improved soft tissue balancing leading ultimately to improved functional outcome. LEVEL OF EVIDENCE: V (expert opinion).

How does laxity after single radius total knee arthroplasty compare with the native knee?

Patients with total knee arthroplasties (TKAs) continue to report dissatisfaction in functional outcome. Stability is a major factor contributing to functionality of TKAs. Implants with single-radius (SR) femoral components are proposed to increase stability throughout the arc of flexion. Using computer navigation and loaded cadaveric legs, we characterized the "envelope of laxity" (EoL) offered by a SR cruciate retaining (CR)-TKA compared with that of the native knee through the arc of flexion in terms of anterior drawer, varus/valgus stress, and internal/external rotation. In both the native knee and the TKA laxity increased with increasing knee flexion. Laxities measured in the three planes of motion were generally comparable between the native knee and TKA from 0° to 110° of flexion. Our results indicate that the SR CR-TKA offers appropriate stability in the absence of soft tissue deficiency.

Clinical biomechanics of instability related to total knee arthroplasty.

BACKGROUND: Tibiofemoral instability is a common reason for total knee arthroplasty failure, and may be attributed to soft tissue deficiency and incorrect ligament balancing. There are many different designs of implant with varying levels of constraint to overcome this instability; however there is little advice for surgeons to assess which is suitable for a specific patient, and soft tissue balance testing during arthroplasty is very subjective. METHOD: The current theories on primary and secondary soft tissue restraints to anterior/posterior, varus/valgus, and internal/external rotational motion of the knee are discussed. The paper reviews biomechanics literature to evaluate instability in the intact and implanted knee. FINDINGS: The paper highlights important intra- and extra-capsular structures in the knee and describes the techniques used by clinicians to assess instability perioperatively. In vitro cadaveric studies were found to be a very useful tool in comparing different implants and contributions of different soft tissues. INTERPRETATION: In vitro cadaveric studies can be utilised in helping less experienced surgeons with soft tissue releases and determining the correct implant. For this to happen, more biomechanical studies must be done to show the impact of release sequences on implanted cadavers, as well as determining if increasingly constrained implants restore the stability of the knee to pre-deficient conditions.

Encapsulation and culture of mammalian cells including corneal cells in alginate hydrogels.

The potential of cell therapy for the regeneration of diseased and damaged tissues is now widely -recognized. As a consequence there is a demand for the development of novel systems that can deliver cells to a particular location, maintaining viability, and then degrade at a predictable rate to release the cells into the surrounding tissues. Hydrogels have attracted much attention in this area, as the hydrogel structure provides an environment that is akin to that of the extracellular matrix. One widely investigated hydrogel is alginate, which has been used for cell encapsulation for more than 30 years. Alginate gels have the potential to be used as 3D cell culture systems and as prosthetic materials, both are applied to regeneration of the cornea. Here, we describe an alginate-based process that has been used for encapsulation of mammalian cells including corneal cells, with high levels of viability, and which allows subsequent retrieval of cell cultures for further characterization.

Calcium-alginate hydrogel-encapsulated fibroblasts provide sustained release of vascular endothelial growth factor.

Vascularization of engineered or damaged tissues is essential to maintain cell viability and proper tissue function. Revascularization of the left ventricle (LV) of the heart after myocardial infarction is particularly important, since hypoxia can give rise to chronic heart failure due to inappropriate remodeling of the LV after death of cardiomyocytes (CMs). Fibroblasts can express vascular endothelial growth factor (VEGF), which plays a major role in angiogenesis and also acts as a chemoattractant and survival factor for CMs and cardiac progenitors. In this in vitro model study, mouse NIH 3T3 fibroblasts encapsulated in 2% w/v Ca-alginate were shown to remain viable for 150 days. Semiquantitative reverse transcription-polymerase chain reaction and immunohistochemistry demonstrated that over 21 days of encapsulation, fibroblasts continued to express VEGF, while enzyme-linked immunosorbent assay showed that there was sustained release of VEGF from the Ca-alginate during this period. The scaffold degraded gradually over the 21 days, without reduction in volume. Cells released from the Ca-alginate at 7 and 21 days as a result of scaffold degradation were shown to retain viability, to adhere to fibronectin in a normal manner, and continue to express VEGF, demonstrating their potential to further contribute to maintenance of cardiac function after scaffold degradation. This model in vitro study therefore demonstrates that fibroblasts encapsulated in Ca-alginate provide sustained release of VEGF.

Alginate hydrogel has a negative impact on in vitro collagen 1 deposition by fibroblasts.

Hydrogels have been widely investigated as 3D culture substrates because of their reported structural similarity to the extracellular matrix (ECM). Limited ECM deposition, however, occurs within these materials, so the resulting "tissues" bear little resemblance to those found in the body. Here matrix deposition by fibroblasts encapsulated within a calcium alginate (Ca-alg) hydrogel was investigated. Although the cells transcribed mRNA for coll Iα over a period of 3 weeks, very little collagen protein deposition was observed within the gel by histology or immunohistochemistry (IHC). Although molecular diffusion demonstrated charge dependency, this did not prevent the flux of both positively and negative charged amino acids through the gel, suggesting that the absence of ECM could not be attributed to substrate limitation. The flux of protein, however, was charge-dependent as proteins with a net negative charge passed quickly through the Ca-alg into the medium. The minimal collagen deposition within the Ca-alg was attributed to a combination of rapid movement of negatively charged procollagen through the gel and steric hindrance of fibril formation.

Cell encapsulation using biopolymer gels for regenerative medicine.

There has been a consistent increase in the mean life expectancy of the population of the developed world over the past century. Healthy life expectancy, however, has not increased concurrently. As a result we are living a larger proportion of our lives in poor health and there is a growing demand for the replacement of diseased and damaged tissues. While traditionally tissue grafts have functioned well for this purpose, the demand for tissue grafts now exceeds the supply. For this reason, research in regenerative medicine is rapidly expanding to cope with this new demand. There is now a trend towards supplying cells with a material in order to expedite the tissue healing process. Hydrogel encapsulation provides cells with a three dimensional environment similar to that experienced in vivo and therefore may allow the maintenance of normal cellular function in order to produce tissues similar to those found in the body. In this review we discuss biopolymeric gels that have been used for the encapsulation of mammalian cells for tissue engineering applications as well as a brief overview of cell encapsulation for therapeutic protein production. This review focuses on agarose, alginate, collagen, fibrin, hyaluronic acid and gelatin since they are widely used for cell encapsulation. The literature on the regeneration of cartilage, bone, ligament, tendon, skin, blood vessels and neural tissues using these materials has been summarised.

Reversible mitotic and metabolic inhibition following the encapsulation of fibroblasts in alginate hydrogels.

Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applications is important in co-culture applications where the growth of one cell type must be inhibited to prevent overgrowth of the scaffold at the expense of another cell type. Also, it is vital for maintaining viability of cells in large constructs before vascularisation occurs. In this study we have shown by means of the Thiazolyl blue (MTT) assay and immuno-staining for proliferating cell nuclear antigen (PCNA) that encapsulating fibroblasts in 2% and 5%w/v calcium-alginate at a density of 7.5 x 10(5)cells/ml as uniformly dispersed entities, enabled cells to maintain viability and caused a reversible mitotic inhibition. Alginate encapsulation also caused reversible metabolic inhibition as demonstrated by the MTT assay and fluorescent staining for mitochondrial membrane potential. Histological evaluation of the alginate constructs containing fibroblasts showed that mitotic and metabolic inhibition was possibly due to cell isolation during the first five weeks of culture. The alginate scaffold degraded with time releasing encapsulated fibroblasts. Upon implantation to a wound site this should ensure that encapsulated cells are able to replace the damaged tissue after sufficient proliferation of the co-cultured cell type or sufficient vascularisation of the construct.

An alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture.

There is significant interest in the development of tissue-engineered skin analogues, which replace both the dermal and the epidermal layer, without the use of animal or human derived products such as collagen or de-epidermalised dermis. In this study, we proposed that alginate hydrogel could be used to encapsulate fibroblasts and that keratinocytes could be cultured on the surface to form a bilayered structure, which could be used to deliver the co-culture to a wound bed, initially providing wound closure and eventually expediting the healing process. Encapsulation of fibroblasts in 2 and 5% w/v alginate hydrogel effectively inhibited their proliferation, whilst maintaining cell viability allowing keratinocytes to grow uninhibited by fibroblast overgrowth to produce a stratified epidermal layer. It was shown that the alginate degradation process was not influenced by the presence of fibroblasts within the hydrogel and that lowering the alginate concentration from 5 to 2% w/v increased the rate of degradation. Fibroblasts released from the scaffold were able to secrete extracellular matrix (ECM) and thus should replace the degrading scaffold with normal ECM following application to the wound site. These findings demonstrate that alginate hydrogel may be an effective delivery vehicle and scaffold for the healing of full-thickness skin wounds.

Impact of cataract surgery on quality of life in patients with early age-related macular degeneration.

PURPOSE: To investigate if cataract surgery improves overall and specific areas of quality of life (QoL) in patients with early age-related macular degeneration (AMD) using the impact of vision impairment (IVI) questionnaire. METHODS: Patients with visually significant cataract and early AMD, who were being considered for cataract surgery in the study eye, were recruited. Eligible patients were randomized to either "early surgery" or "standard surgery" (standard cataract surgery waiting time of 6 months) groups. The IVI, sociodemographic, and clinical data were collected. Rasch analysis was used to estimate QoL person measures at baseline and follow-up. The data were analyzed using repeated measures ANOVA. Effect sizes were calculated using Cohen's d coefficient. RESULTS: Fifty six patients (mean age = 78.5 years and visual acuity = 6/15) had one eye randomly allocated to either the early surgery (n = 29) or standard surgery (n = 27) groups. At follow-up, significant interaction effects were found for the overall IVI score [F(1,54) = 17.7; p < 0.001], the emotional well-being [F(1,54) = 13.4; p = 0.001], mobility and independence [F(1,54) = 13.4; p = 0.001], and reading and accessing information subscales [F(1,54) = 13.1; p = 0.001]. The standard surgery group systematically recorded worse scores at 6 months on all QoL measures whereas the early surgery group recorded significant gains (p < 0.001; Cohen's d = 0.66 to 0.91) on all of them. Visual acuity in the study eye significantly improved in the early surgery group only (Cohen's d = 1.1; p < 0.05) and improvement in log MAR lines read was identified as the single independent predictor of enhanced QoL explaining between 26 and 34% of the variance in the IVI scores. CONCLUSIONS: Cataract surgery is justified in patients with early AMD. It brings significant improvements in visual acuity, aspects of daily living, and overall QoL.

Analysis of the Y402H variant of the complement factor H gene in age-related macular degeneration.

PURPOSE: Recent studies in U.S. populations have indicated that the Y402H variant of the complement factor H (CFH) gene contains a major risk susceptibility allele for age-related macular degeneration (AMD). This study was conducted to ascertain whether this is also true in a non-U.S. population and also whether the at-risk allele is associated with the clinical phenotype of disease and the age at diagnosis. METHODS: Two hundred thirty-six unrelated individuals with AMD and 144 unrelated but ethnically matched control subjects were recruited and examined. All subjects completed a standard questionnaire, were given a fundus examination, and provided a blood sample for DNA extraction. Alleles of Y402H in the CFH gene were determined by use of a MALDI-TOF-based approach followed by statistical analysis. RESULTS: Individuals with AMD who had at least one copy of the C allele of Y402H had an increased risk of disease (odds ratio [OR] 2.98; 95% confidence interval [CI] 1.81-4.93) compared with cases with the T allele. On subgroup analysis, this risk was found to be most significant in individuals with neovascular disease (OR 4.34; 95% CI 1.94, 9.71). In addition, individuals with neovascular disease who were homozygous CC presented with a significant 7.0-year earlier age at diagnosis relative to those individuals who were homozygous TT. The population-attributable risk for the C allele ranged between 47% to 69%, depending on the AMD disease subtype. CONCLUSIONS: The C allele of Y402H represents a significant risk factor in individuals with AMD, and this effect is most pronounced in individuals with neovascular disease.

Genetic variation in BEACON influences quantitative variation in metabolic syndrome-related phenotypes.

The BEACON gene (also known as UBL5) was identified as differentially expressed between lean and obese Psammomys obesus, a polygenic animal model of obesity, type 2 diabetes, and dyslipidemia. The human homologue of BEACON is located on chromosome 19p, a region likely to contain genes affecting metabolic syndrome-related quantitative traits as established by linkage studies. To assess whether the human BEACON gene may be involved in influencing these traits, we exhaustively analyzed the complete gene for genetic variation in 40 unrelated individuals and identified four variants (three novel). The two more common variants were tested for association with a number of quantitative metabolic syndrome-related traits in two large cohorts of unrelated individuals. Significant associations were found between these variants and fat mass (P = 0.026), percentage of fat (P = 0.001), and waist-to-hip ratio (P = 0.031). The same variants were also associated with total cholesterol (P = 0.024), LDL cholesterol (P = 0.019), triglycerides (P = 0.006), and postglucose load insulin levels (P = 0.018). Multivariate analysis of these correlated phenotypes also yielded a highly significant association (P = 0.0004), suggesting that BEACON may influence phenotypic variation in metabolic syndrome-related traits.