Enthesis tissue engineering: biological requirements meet at the interface

Tendon-to-bone interface (enthesis) exhibits a complex multiscale architectural and compositional organization maintained by a heterogeneous cellular environment. Orthopedic surgeons have been facing several challenges when treating tendon pullout or tear from the bony insertion due to unsatisfactory surgical outcomes and high retear rates. The limited understanding of enthesis hinders the development of new treatment options toward enhancing regeneration. Mimicking the natural tissue structure and composition is still a major challenge to be overcome. In this review, we critically assess current tendon-to-bone interface tissue engineering strategies through the use of biological, biochemical, or biophysical cues, which must be ultimately combined into sophisticated gradient systems. Cellular strategies are described, focusing on cell sources and cocultures to emulate a physiological heterotypic niche, as well as hypoxic environments, alongside with growth factor delivery and the use of platelet-rich hemoderivatives. Biomaterial design considerations are revisited, highlighting recent progresses in tendon-to-bone scaffolds. Mechanical loading is addressed to uncover prospective engineering advances. Finally, research challenges and translational aspects are considered. In summary, we highlight the importance of deeply investigating enthesis biology toward establishing foundational expertise and integrate cues from the native niche into novel biomaterial engineering, aiming at moving today's research advances into tomorrow's regenerative therapies.Authors thank the support from the European Union Framework Programme for Research and Innovation HORIZON2020 [TEAMING Grant agreement No 739572 - The Discoveries CTR]; FCT–Fundação para a Ciência e a Tecnologia for the PhD grant of IC [PD/BD/128088/2016]; the Project NORTE-01-0145-FEDER-000021:“Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marine-derived biomaterials and stem cells”, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and the ERC Consolidator grant of ME [ERC-2017-CoG-772817]

Repeatability and Reproducibility of Retinal Thickness Measurements With Spectral-Domain Optical Coherence Tomography Using Different Scan Parameters

PURPOSE: To evaluate repeatability (test-retest) and reproducibility of retinal thickness measurements using spectral-domain optical coherence tomography with different densities of A-scans per B-scan and different frames per B-scan for real-time averaging. METHODS: Twelve healthy subjects were analyzed by spectral-domain optical coherence tomography. Raster lines analysis with 19 B-scans over the examined area centered on the macula was performed. Images were acquired both in standard-density (768 A-scans/B-scan) and high-density (1,536 A-scans/B-scan) modalities. Moreover, images were acquired using 2 and 20 frames/B-scan for real-time averaging. Each analysis was repeated twice to test for repeatability. RESULTS: Intersession repeatability was good for all studied analysis protocols, with Lin concordance correlation coefficient values ranging between 0.88 and 1.00. Reproducibility assessment showed consistent retinal thickness measurements using variable scan density, with Bland-Altman limits of agreement of -6 \u3bcm to 6 \u3bcm in the central area. Reproducibility assessment showed consistent retinal thickness measurements using different number of frames used in the real-time averaging process, with Bland-Altman limits of agreement of -8 \u3bcm to 4 \u3bcm in the central area. CONCLUSION: Spectral-domain optical coherence tomography is a very reliable tool for central retinal thickness assessment. Changes in the number of A-scans/B-scan and in frames used for real-time averaging do not affect repeatability and reproducibility

RPE tears in wet AMD: Natural history vs a surgical approach

N/

The dark atrophy with indocyanine green angiography in Stargardt disease

PURPOSE: To evaluate differences in fluorescein angiography (FA) and indocyanine green angiography (ICGA), findings between subjects affected by Stargardt disease (STGD) and atrophic AMD. METHODS: This was a consecutive, cross-sectional case series. A total of 24 eyes of 12 patients with STGD and 23 eyes of 14 patients with atrophic AMD were enrolled in the study. Patients underwent dynamic simultaneous FA and ICGA using a dual beam confocal scanning system. Images were recorded from the initial filling of choroidal and retinal vessels throughout all the phases of the angiogram. Spectral-domain optical coherence tomography (SD-OCT) and fundus autofluorescence were also executed. FA and ICGA findings in the two groups were evaluated. RESULTS: In 92% (22/24) of eyes affected by STGD, ICGA showed hypocyanescence from the areas of atrophy, more evident in the late phases. This finding, defined as ICGA-imaged "dark atrophy," was present in only 13% (3/23) of the eyes affected by atrophic AMD. The remaining eyes in both groups showed iso- or mild hypercyanescence from the areas of atrophy. Eyes with ICGA-imaged dark atrophy, both in STGD and in atrophic AMD groups, did not show early obscuration of the choroidal vessels by FA. SD-OCT revealed morphologically intact choroid in STGD patients with ICGA-imaged dark atrophy. In atrophic AMD eyes with ICGA-imaged dark atrophy, SD-OCT revealed a severely thinned choroid. CONCLUSIONS: Hypocyanescence by ICGA from the areas of atrophy was more frequent in STGD compared with atrophic AMD. This finding, along with SD-OCT evidence of intact choroid, suggests a possible selective damage of the choriocapillaris in STGD

P-635 The impact of force-degraded variants of recombinant human follicle stimulating hormone alfa (r-hFSH alfa) on in-vitro and in-vivo biological activity

Study question: Can an in-vitro bioassay assess the potency of r-hFSH-alfa force-degraded variants with similar ability to the in-vivo rat bioassay described in EU Pharmacopoeia (EU-Pharm 2285) ? Summary answer: The in-vitro bioassay showed similar ability to the in-vivo bioassay for estimating the impact of r-hFSH-alfa variants, resulting from process-related modifications, on biological activity

P-635 The impact of force-degraded variants of recombinant human follicle stimulating hormone alfa (r-hFSH alfa) on <i>in-vitro</i> and <i>in-vivo</i> biological activity

Abstract Study question Can an in-vitro bioassay assess the potency of r-hFSH-alfa force-degraded variants with similar ability to the in-vivo rat bioassay described in EU Pharmacopoeia (EU-Pharm 2285)? Summary answer The in-vitro bioassay showed similar ability to the in-vivo bioassay for estimating the impact of r-hFSH-alfa variants, resulting from process-related modifications, on biological activity. What is known already The active ingredient of Gonal f® (Merck KGaA), follitropin-alfa (INN, r-hFSH-alfa), mimics the action of endogenous FSH by binding to, and subsequently activating, the hFSH-receptor (FSH-R), regulating cellular metabolism and oocyte survival/maturation. Structural modifications in r-hFSH-alfa glycosylation, oxidation, or other biochemical changes, may occur during the r-hFSH-alfa manufacturing process and may impact its efficacy/safety. The rat in-vivo bioassay (EU Pharmacopeia) is routinely used to assess r-hFSH-alfa potency by measuring ovarian weight increase. We evaluated whether an in-vitro bioassay has the same ability as the rat in-vivo bioassay to detect changes in biological activity caused by r-hFSH-alfa structural modifications. Study design, size, duration Three r-hFSH-alfa (Gonal f®) batches were stressed under eight chemical/physical/enzymatic treatments. The resulting degraded samples were compared with untreated samples using in-vivo and in-vitro bioassays. Participants/materials, setting, methods Force-degraded r-hFSH-alfa variants were produced by chromatographic separation (acidic/basic enrichment), chemical/physical stress (acidic/basic pH incubation, thermal/oxidative stress) and enzymatic treatments (de-galactosylation and de-sialylation). r-hFSH-alfa potency was measured via a rat in-vivo bioassay (European Pharmacopoeia 2285), assessing ovarian weight increase, and an in-vitro bioassay (cell line expressing the transmembrane hFSH-R), assessing cell-specific metabolic cascade. Force-degraded and untreated samples were compared via analysis of variance. Main results and the role of chance All force-degraded samples were characterized by the chemical/physical analyses panel for quality control. r-hFSH-alfa forced degradation modified the critical quality attributes of the samples; namely, increased the presence of r-hFSH-alfa oxidized forms and/or free subunits up to 50% above product specification, and modified the sialic acid level across variants, generating significantly more hypo-sialylated forms when compared with the untreated control samples. The increase in r-hFSH-alfa free subunits significantly reduced r-h-FSH-alfa biological activity compared with untreated samples, in both the in-vivo and in-vitro assays (p &amp;lt; 0.001 for both). The increase in r-hFSH-alfa oxidized forms significantly reduced r-hFSH-alfa biological activity in-vitro (p &amp;lt;0.001) and, to a lesser extent, in-vivo (p &amp;lt;0.006). A gradual decrease in r-hFSH-alfa sialylation decreased r-hFSH-alfa biological activity in-vivo, indicating a second-order polynomial correlation, and increased r-hFSH-alfa biological activity in-vitro, indicating a negative linear correlation (slope significance p&amp;lt;0.001, R2=0.996). De-sialylation reduces r-hFSH-alfa steric hindrance during the interaction of r-hFSH-alfa and the FSH-R, increasing both r-hFSH-alfa–FSH-R affinity and r-hFSH-alfa biological activity in-vitro, while increasing the rate of r-hFSH-alfa metabolism, resulting in decreased r-hFSH-alfa biological activity in-vivo. Both assays showed a similar ability to identify differences in critical quality attribute levels (sialylation, oxidation, free-subunits), with the intent to identify out-of-specification batches. Limitations, reasons for caution r-hFSH-alfa forced degradation produced more than one structural/chemical modification in most variants (except for de-sialylation); therefore, the effect of discrete modifications could not be studied. Additional studies are needed to show that in-vitro methods can ultimately replace in-vivo bioassays to identify differences in critical quality attribute levels during r-hFSH-alfa manufacturing. Wider implications of the findings Chemical and/or structural modifications of r-hFSH-alfa strongly impact r-hFSH-alfa biological activity and related potency. The development of an in-vitro bioassay for the accurate measurement of r-hFSH-alfa potency may serve to replace the in-vivo bioassay, according to EMA indications (CPMP/SWP728/95). Trial registration number not applicable </jats:sec