Considering the cellular landscape in marrow stimulation techniques for cartilage repair.

BACKGROUND: Marrow stimulation is a common reparative approach to treat injuries to cartilage and other soft tissues (e.g., rotator cuff). It involves the recruitment of bone marrow elements and mesenchymal stem cells (MSCs) into the defect, theoretically initiating a regenerative process. However, the resulting repair tissue is often weak and susceptible to deterioration with time. The populations of cells at the marrow stimulation site (beyond MSCs), and their contribution to inflammation, vascularity, and fibrosis, may play a role in quality of the repair tissue. SUMMARY: In this review, we accomplish three goals: 1) systematically review clinical trials on the augmentation of marrow stimulation and evaluate their assumptions on the biological elements recruited; 2) detail the cellular populations in bone marrow and their impact on healing; and 3) highlight emerging technologies and approaches that could better guide these specific cell populations towards enhanced cartilage or soft tissue formation. KEY MESSAGES: We found that most clinical trials do not account for cell heterogeneity, nor do they specify the regenerative element recruited, and those that do typically utilize descriptions such as "clots", "elements", and "blood". Furthermore, our review of bone marrow cell populations demonstrates a dramatically heterogenous cell population, including hematopoietic cells, immune cells, fibroblasts, macrophages, and only a small population of MSCs. Finally, the field has developed numerous innovative techniques to enhance the chondrogenic potential (and reduce the anti-regenerative impacts) of these various cell types. We hope this review will guide approaches that account for cellular heterogeneity and improve marrow stimulation techniques to treat chondral defects.

A cholinergic basal forebrain feeding circuit modulates appetite suppression.

Atypical food intake is a primary cause of obesity and other eating and metabolic disorders. Insight into the neural control of feeding has previously focused mainly on signalling mechanisms associated with the hypothalamus, the major centre in the brain that regulates body weight homeostasis. However, roles of non-canonical central nervous system signalling mechanisms in regulating feeding behaviour have been largely uncharacterized. Acetylcholine has long been proposed to influence feeding owing in part to the functional similarity between acetylcholine and nicotine, a known appetite suppressant. Nicotine is an exogenous agonist for acetylcholine receptors, suggesting that endogenous cholinergic signalling may play a part in normal physiological regulation of feeding. However, it remains unclear how cholinergic neurons in the brain regulate food intake. Here we report that cholinergic neurons of the mouse basal forebrain potently influence food intake and body weight. Impairment of cholinergic signalling increases food intake and results in severe obesity, whereas enhanced cholinergic signalling decreases food consumption. We found that cholinergic circuits modulate appetite suppression on downstream targets in the hypothalamus. Together our data reveal the cholinergic basal forebrain as a major modulatory centre underlying feeding behaviour.

Personalized Fiber-Reinforcement Networks for Meniscus Reconstruction.

The menisci are fibrocartilaginous tissues that are crucial to the load-sharing and stability of the knee, and when injured, these properties are compromised. Meniscus replacement scaffolds have utilized the circumferential alignment of fibers to recapitulate the microstructure of the native meniscus; however, specific consideration of size, shape, and morphology has been largely overlooked. The purpose of this study was to personalize the fiber-reinforcement network of a meniscus reconstruction scaffold. Human cadaveric menisci were measured for a host of tissue (length, width) and subtissue (regional widths, root locations) properties, which all showed considerable variability between donors. Next, the asymmetrical fiber network was optimized to minimize the error between the dimensions of measured menisci and predicted fiber networks, providing a 51.0% decrease (p = 0.0091) in root-mean-square (RMS) error. Finally, a separate set of human cadaveric knees was obtained, and donor-specific fiber-reinforced scaffolds were fabricated. Under cyclic loading for load-distribution analysis, in situ implantation of personalized scaffolds following total meniscectomy restored contact area (253.0 mm2 to 488.9 mm2, p = 0.0060) and decreased contact stress (1.96 MPa to 1.03 MPa, p = 0.0025) to near-native values (597.4 mm2 and 0.83 MPa). Clinical use of personalized meniscus devices that restore physiologic contact stress distributions may prevent the development of post-traumatic osteoarthritis following meniscal injury.

Carbodiimide cross-linking counteracts the detrimental effects of gamma irradiation on the physical properties of collagen-hyaluronan sponges.

Collagen-based scaffolds are extensively used in biomaterials and tissue engineering applications. These scaffolds have shown great biocompatibility and versatility, but their relatively low mechanical properties may limit use in orthopaedic load-bearing applications. Moreover, terminal sterilization with gamma irradiation, as is commonly performed with commercial devices, presents concerns over structural integrity and enzymatic stability. Therefore, the goal of this study was to test the hypothesis that EDC/NHS cross-linking (10 mM/5 mM) can protect collagen-hyaluronan sponges from the damaging effects of gamma irradiation. Specifically, we evaluated compressive and tensile mechanical properties, enzymatic stability, porosity and pore size, and swelling ratio. Ultimate tensile strength and elastic modulus exhibited increases (168.5 and 245.8%, respectively) following irradiation, and exhibited over tenfold increases (1049.2 and 1270.6%, respectively) following cross-linking. Irradiation affected pore size (38.4% decrease), but cross-linking prior to irradiation resulted in only a 17.8% decrease. Cross-linking also showed an offsetting effect on the equilibrium modulus, enzymatic stability, and swelling ratio of sponges. These results suggest that carbodiimide cross-linking of collagen-hyaluronan sponges can mitigate the structural damage typically experienced during gamma irradiation, warranting their use in tissue engineering applications.

Prediction of Hydrolysis Products of Organic Chemicals under Environmental pH Conditions.

Cheminformatics-based software tools can predict the molecular structure of transformation products using a library of transformation reaction schemes. This paper presents the development of such a library for abiotic hydrolysis of organic chemicals under environmentally relevant conditions. The hydrolysis reaction schemes in the library encode the process science gathered from peer-reviewed literature and regulatory reports. Each scheme has been ranked on a scale of one to six based on the median half-life in a data set compiled from literature-reported hydrolysis rates. These ranks are used to predict the most likely transformation route when more than one structural fragment susceptible to hydrolysis is present in a molecule of interest. Separate rank assignments are established for pH 5, 7, and 9 to represent standard conditions in hydrolysis studies required for registration of pesticides in Organisation for Economic Co-operation and Development (OECD) member countries. The library is applied to predict the likely hydrolytic transformation products for two lists of chemicals, one representative of chemicals used in commerce and the other specific to pesticides, to evaluate which hydrolysis reaction pathways are most likely to be relevant for organic chemicals found in the natural environment.

Impact of a pharmacist-led oral chemotherapy-monitoring program in patients with metastatic castrate-resistant prostate cancer.

BACKGROUND: With the introduction of oral chemotherapy, the paradigm for cancer treatment is shifting. Use of oral chemotherapy agents offers a non-invasive option for patients with metastatic castrate-resistant prostate cancer. However, these medications are not without challenges including strict adherence for optimal effects, novel toxicity profiles, frequent lab parameter monitoring, high cost, and proper handling and disposal methods. Pharmacists are positioned to play a key role in providing patients with the education required to assure an optimal treatment course is carried out. METHODS: Two cohorts of patients receiving abiraterone, bicalutamide, or enzalutamide for metastatic castrate-resistant prostate cancer seen in our outpatient cancer center 21 months before and 24 months after the implementation of a pharmacist-led oral chemotherapy-monitoring program in December of 2012 were retrospectively compared. Patients were evaluated for number of interventions, adherence to lab parameter monitoring, and overall time on each therapy. RESULTS: Of the 64 patients identified, 31 patients fulfilled inclusion criteria. A significant increase in the average number of interventions per patient (6.9 vs. 2.6; P = 0.004) and adherence to lab parameter monitoring (10 vs. 3; P = 0.04) in the post-program implementation cohort was found. However, no significant difference in overall time on therapy (10.3 vs. 8.1; P = 0.341) between the two groups was observed. CONCLUSION: These results suggest a potential opportunity exists to maximize oral chemotherapy treatment outcomes with the addition of a formalized monitoring program directed by an oncology pharmacist.

Apcdd1 stimulates oligodendrocyte differentiation after white matter injury.

Wnt signaling plays an essential role in developmental and regenerative myelination of the CNS, therefore it is critical to understand how the factors associated with the various regulatory layers of this complex pathway contribute to these processes. Recently, Apcdd1 was identified as a negative regulator of proximal Wnt signaling, however its role in oligodendrocyte (OL) differentiation and reymelination in the CNS remain undefined. Analysis of Apcdd1 expression revealed dynamic expression during OL development, where its expression is upregulated during differentiation. Functional studies using ex vivo and in vitro OL systems revealed that Apcdd1 promotes OL differentiation, suppresses Wnt signaling, and associates with ß-catenin. Application of these findings to white matter injury (WMI) models revealed that Apcdd1 similarly promotes OL differentiation after gliotoxic injury in vivo and acute hypoxia ex vivo. Examination of Apcdd1 expression in white matter lesions from neonatal WMI and adult multiple sclerosis revealed its expression in subsets of oligodendrocyte (OL) precursors. These studies describe, for the first time, the role of Apcdd1 in OLs after WMI and reveal that negative regulators of the proximal Wnt pathway can influence regenerative myelination, suggesting a new therapeutic strategy for modulating Wnt signaling and stimulating repair after WMI.

Mutation of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase at Trp-110 affects stereoselectivity of aromatic ketone reduction.

Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP(+))-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.

Revealing Early Spatial Patterns of Cellular Responsivity in Fiber-Reinforced Microenvironments.

Fiber-reinforcement approaches have been used to replace aligned tissues with engineered constructs after injury or surgical resection, strengthening soft biomaterial scaffolds and replicating anisotropic, load-bearing properties. However, most studies focus on the macroscale aspects of these scaffolds, rarely considering the cell-biomaterial interactions that govern remodeling and extracellular matrix organization toward aligned neo-tissues. As initial cell-biomaterial responses within fiber-reinforced microenvironments likely influence the long-term efficacy of repair and regeneration strategies, here we elucidate the roles of spatial orientation, substrate stiffness, and matrix remodeling on early cell-fiber interactions. Bovine mesenchymal stromal cells (MSCs) were cultured in soft fibrin gels reinforced with a stiff 100 µm polyglycolide-co-caprolactone fiber. Gel stiffness and remodeling capacity were modulated by fibrinogen concentration and aprotinin treatment, respectively. MSCs were imaged at 3 days and evaluated for morphology, mechanoresponsiveness (nuclear Yes-associated protein [YAP] localization), and spatial features including distance and angle deviation from fiber. Within these constructs, morphological conformity decreased as a function of distance from fiber. However, these correlations were weak (R2 = 0.01043 for conformity and R2 = 0.05542 for nuclear YAP localization), illustrating cellular heterogeneity within fiber-enforced microenvironments. To better assess cell-fiber interactions, we applied machine-learning strategies to our heterogeneous dataset of cell-shape and mechanoresponsive parameters. Principal component analysis (PCA) was used to project 23 input parameters (not including distance) onto 5 principal components (PCs), followed by agglomerative hierarchical clustering to classify cells into 3 groups. These clusters exhibited distinct levels of morpho-mechanoresponse (combination of morphological conformity and YAP signaling) and were classified as high response (HR), medium response (MR), and low response (LR) clusters. Cluster distribution varied spatially, with most cells (61%) closest to the fiber (0-75 µm) belonging to the HR cluster, and most cells (55%) furthest from the fiber (225-300 µm) belonging to the LR cluster. Modulation of gel stiffness and fibrin remodeling showed differential effects for HR cells, with stiffness influencing the level of mechanoresponse and remodeling capacity influencing the location of responding cells. Together, these novel findings demonstrate early trends in cellular patterning of the fiber-reinforced microenvironment, showing how spatial orientation, substrate biophysical properties, and matrix remodeling may guide the amplitude and localization of cellular mechanoresponses. These trends may guide approaches to optimize the design of microscale scaffold architecture and substrate properties for enhancing organized tissue assembly at the macroscale.

Automated quantitative assessment of bone contusions and overlying articular cartilage following anterior cruciate ligament injury.

Quantitative methods to characterize bone contusions and associated cartilage injury remain limited. We combined standardized voxelwise normalization and 3D mapping to automate bone contusion segmentation post-anterior cruciate ligament (ACL) injury and evaluate anomalies in articular cartilage overlying bone contusions. Forty-five patients (54% female, 26.4 ± 11.8 days post-injury) with an ACL tear underwent 3T magnetic resonance imaging of their involved and uninvolved knees. A novel method for voxelwise normalization and 3D anatomical mapping was used to automate segmentation, labeling, and localization of bone contusions in the involved knee. The same mapping system was used to identify the associated articular cartilage overlying bone lesions. Mean regional T1ρ was extracted from articular cartilage regions in both the involved and uninvolved knees for quantitative paired analysis against ipsilateral cartilage within the same compartment outside of the localized bone contusion. At least one bone contusion lesion was detected in the involved knee within the femur and/or tibia following ACL injury in 42 participants. Elevated T1ρ (p = 0.033) signal were documented within the articular cartilage overlying the bone contusions resulting from ACL injury. In contrast, the same cartilaginous regions deprojected onto the uninvolved knees showed no ipsilateral differences (p = 0.795). Automated bone contusion segmentation using standardized voxelwise normalization and 3D mapping deprojection identified altered cartilage overlying bone contusions in the setting of knee ACL injury.

Mechanical activation of a multimeric adhesive protein through domain conformational change.

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF's crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

Transient inhibition of meniscus cell migration following acute inflammatory challenge.

Meniscus tears represent a common orthopedic injury that often requires surgery to restore pain-free function. The need for surgical intervention is due, in part, to the inflammatory and catabolic environment that inhibits meniscus healing after injury. In other organ systems, healing is dependent on the migration of cells to the site of injury; however, in the meniscus, it is currently unknown how the microenvironment dictates cell migration in the postinjury inflamed setting. Here, we investigated how inflammatory cytokines alter meniscal fibrochondrocyte (MFC) migration and sensation of microenvironmental stiffness. We further tested whether an FDA approved interleukin-1 receptor antagonist (IL-1Ra; Anakinra) could rescue migratory deficits caused by inflammatory challenge. When cultured in the presence of inflammatory cytokines (tumor necrosis factor-α [TNF-α] or interleukin-1ß [IL-1ß]) for 1 day, MFC migration was inhibited for 3 days before returning to control levels at Day 7. This migratory deficit was clear in three-dimensional as well, where fewer MFCs exposed to inflammatory cytokines migrated from a living meniscal explant compared with control. Notably, addition of IL-1Ra to MFCs previously exposed to IL-1ß restored migration to baseline levels. This study demonstrates that joint inflammation can have negative impacts on meniscus cell migration and mechanosensation, affecting their potential for repair, and that resolution of this inflammation with concurrent anti-inflammatories can reverse these deficits. Future work will apply these findings to mitigate the negative consequences of joint inflammation and promote repair in a clinically relevant meniscus injury model.

Improved Cartilage Protection with Low Molecular Weight Hyaluronic Acid Hydrogel.

Traumatic joint injuries are common, leading to progressive tissue degeneration and the development of osteoarthritis. The post-traumatic joint experiences a pro-inflammatory milieu, initiating a subtle but deteriorative process in cartilage tissue. To prevent or even reverse this process, our group previously developed a tissue-penetrating methacrylated hyaluronic acid (MeHA) hydrogel system, crosslinked within cartilage to restore and/or protect the tissue. In the current study, we further optimized this approach by investigating the impact of biomaterial molecular weight (MW; 20, 75, 100 kDa) on its integration within and reinforcement of cartilage, as well as its ability to protect tissue degradation in a catabolic state. Indeed, the low MW MeHA integrated and reinforced cartilage tissue better than the high MW counterparts. Furthermore, in a 2 week IL-1ß explant culture model, the 20 kDa MeHA demonstrated the most protection from biphasic mechanical loss, best retention of proteoglycans (Safranin O staining), and least aggrecan breakdown (NITEGE). Thus, the lower MW MeHA gels integrated better into the tissue and provided the greatest protection of the cartilage matrix. Future work will test this formulation in a preclinical model, with the goal of translating this therapeutic approach for cartilage preservation.

Exogenous All-Trans Retinoic Acid Induces Myopia and Alters Scleral Biomechanics in Mice.

Purpose: Ocular all-trans retinoic acid (atRA) levels are influenced by visual cues, and exogenous atRA has been shown to increase eye size in chickens and guinea pigs. However, it is not clear whether atRA induces myopic axial elongation via scleral changes. Here, we test the hypothesis that exogenous atRA will induce myopia and alter scleral biomechanics in the mouse. Methods: Male C57BL/6J mice were trained to voluntarily ingest atRA + vehicle (1% atRA in sugar, 25 mg/kg) (RA: n = 16 animals) or vehicle only (Ctrl: n = 14 animals). Refractive error (RE) and ocular biometry were measured at baseline and after 1 and 2 weeks of daily atRA treatment. Eyes were used in ex vivo assays to measure scleral biomechanics (unconfined compression: n = 18), total scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 23), and specific sGAGs (immunohistochemistry: n = 18). Results: Exogenous atRA caused myopic RE and larger vitreous chamber depth (VCD) to develop by 1 week (RE: -3.7 ± 2.2 diopters [D], P < 0.001; VCD: +20.7 ± 15.1 µm, P < 0.001), becoming more severe by 2 weeks (RE: -5.7 ± 2.2 D, P < 0.001; VCD: +32.3 ± 25.8 µm, P < 0.001). The anterior eye biometry was unaffected. While scleral sGAG content was not measurably affected, scleral biomechanics were significantly altered (tensile stiffness: -30% ± 19.5%, P < 0.001; permeability: +60% ± 95.3%, P < 0.001). Conclusions: In mice, atRA treatment results in an axial myopia phenotype. Eyes developed myopic RE and larger VCD without the anterior eye being affected. The decrease in stiffness and increase in permeability of the sclera are consistent with the form-deprivation myopia phenotype.

Cartilage-penetrating hyaluronic acid hydrogel preserves tissue content and reduces chondrocyte catabolism.

Articular cartilage injuries have a limited healing capacity and, due to inflammatory and catabolic activities, often experience progressive degeneration towards osteoarthritis. Current repair techniques generally provide short-term symptomatic relief; however, the regeneration of hyaline cartilage remains elusive, leaving both the repair tissue and surrounding healthy tissue susceptible to long-term wear. Therefore, methods to preserve cartilage following injury, especially from matrix loss and catabolism, are needed to delay, or even prevent, the deteriorative process. The goal of this study was to develop and evaluate a cartilage-penetrating hyaluronic-acid (HA) hydrogel to improve damaged cartilage biomechanics and prevent tissue degeneration. At time zero, the HA-based hydrogel provided a 46.5% increase in compressive modulus and a decrease in permeability after simulated degeneration of explants (collagenase application). Next, in a degenerative culture model (interleukin-1ß [IL-1ß] for 2 weeks), hydrogel application prior to or midway through the culture mitigated detrimental changes to compressive modulus and permeability observed in non-treated explants. Furthermore, localized loss of proteoglycan was observed in degenerative culture conditions alone (non-treated), but hydrogel administration significantly improved the retention of matrix elements. Finally, NITEGE staining and gene expression analysis showed the ability of the HA gel to decrease chondrocyte catabolic activity. These results highlight the importance of reinforcing damaged cartilage with a biomaterial system to both preserve tissue content and reduce catabolism associated with injury and inflammation.

Plant Tissue Parenchyma and Vascular Bundles Selectively Regulate Stem Cell Mechanosensing and Differentiation.

Introduction: Plant tissues are plentiful, diverse, and due to convergent evolution are structurally similar to many animal tissues. Decellularized plant tissues feature microtopographies that resemble cancellous bone (porous parenchyma) and skeletal muscle (fibrous vascular bundles). However, the use of plant tissues as an inexpensive and abundant biomaterial for controlling stem cell behavior has not been widely explored. Methods: Celery plant tissues were cut cross-sectionally (porous parenchyma) or longitudinally (fibrous vascular bundles) and decellularized. Human mesenchymal stem cells (MSCs) were then cultured atop plant tissues and confocal imaging of single cells was used to evaluate the early effects of microtopography on MSC adhesion, morphology, cytoskeletal alignment, Yes-associated protein (YAP) signaling, and downstream lineage commitment to osteogenic or myogenic phenotypes. Results: Microtopography was conserved post plant tissue decellularization and MSCs attached and proliferated on plant tissues. MSCs cultured on porous parenchyma spread isotropically along the periphery of plant tissue pores. In contrast, MSCs cultured on vascular bundles spread anisotropically and aligned in the direction of fibrous vascular bundles. Differences in microtopography also influenced MSC nuclear YAP localization and actin anisotropy, with higher values observed on fibrous tissues. When exposed to osteogenic or myogenic culture medium, MSCs on porous parenchyma had a higher percentage of cells stain positive for bone biomarker alkaline phosphatase, whereas myoblast determination protein 1 (MyoD) was significantly upregulated for MSCs on fibrous vascular bundles. Conclusions: Together, these results show that plant tissues are an abundant biomaterial with defined microarchitecture that can reproducibly regulate MSC morphology, mechanosensing, and differentiation. Supplementary Information: The online version of this article contains supplementary material available 10.1007/s12195-022-00737-9.

Stable human cartilage progenitor cell line stimulates healing of meniscal tears and attenuates post-traumatic osteoarthritis.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine.

Altered Structure and Function of Murine Sclera in Form-Deprivation Myopia.

Purpose: The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process. Methods: Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE + choroid + sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes). Results: Significant myopia developed after 1 (-2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (-4.1 ± 0.7 D, P = 0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = -31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week. Conclusions: We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

In Situ Assessment of Porcine Osteochondral Repair Tissue in the Visible-Near Infrared Spectral Region.

Standard assessment of cartilage repair progression by visual arthroscopy can be subjective and may result in suboptimal evaluation. Visible-near infrared (Vis-NIR) fiber optic spectroscopy of joint tissues, including articular cartilage and subchondral bone, provides an objective approach for quantitative assessment of tissue composition. Here, we applied this technique in the 350-2,500 nm spectral region to identify spectral markers of osteochondral tissue during repair with the overarching goal of developing a new approach to monitor repair of cartilage defects in vivo. Full thickness chondral defects were created in Yucatan minipigs using a 5-mm biopsy punch, and microfracture (MFx) was performed as a standard technique to facilitate repair. Tissues were evaluated at 1 month (in adult pigs) and 3 months (in juvenile pigs) post-surgery by spectroscopy and histology. After euthanasia, Vis-NIR spectra were collected in situ from the defect region. Additional spectroscopy experiments were carried out in vitro to aid in spectral interpretation. Osteochondral tissues were dissected from the joint and evaluated using the conventional International Cartilage Repair Society (ICRS) II histological scoring system, which showed lower scores for the 1-month than the 3-month repair tissues. In the visible spectral region, hemoglobin absorbances at 540 and 570 nm were significantly higher in spectra from 1-month repair tissue than 3-month repair tissue, indicating a reduction of blood in the more mature repair tissue. In the NIR region, we observed qualitative differences between the two groups in spectra taken from the defect, but differences did not reach significance. Furthermore, spectral data also indicated that the hydrated environment of the joint tissue may interfere with evaluation of tissue water absorbances in the NIR region. Together, these data provide support for further investigation of the visible spectral region for assessment of longitudinal repair of cartilage defects, which would enable assessment during routine arthroscopy, particularly in a hydrated environment.