Differential Efficacy of Small Molecules Dynasore and Mdivi-1 for the Treatment of Dry Eye Epitheliopathy or as a Countermeasure for Nitrogen Mustard Exposure of the Ocular Surface.

The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.

Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells.

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Dynasore Protects Corneal Epithelial Cells Subjected to Hyperosmolar Stress in an In Vitro Model of Dry Eye Epitheliopathy.

Epitheliopathy at the ocular surface is a defining sign of dry eye disease, a common disorder that affects 10% to 30% of the world's population. Hyperosmolarity of the tear film is one of the main drivers of pathology, with subsequent endoplasmic reticulum (ER) stress, the resulting unfolded protein response (UPR), and caspase-3 activation implicated in the pathway to programmed cell death. Dynasore, is a small molecule inhibitor of dynamin GTPases that has shown therapeutic effects in a variety of disease models involving oxidative stress. Recently we showed that dynasore protects corneal epithelial cells exposed to the oxidant tBHP, by selective reduction in expression of CHOP, a marker of the UPR PERK branch. Here we investigated the capacity of dynasore to protect corneal epithelial cells subjected to hyperosmotic stress (HOS). Similar to dynasore's capacity to protect against tBHP exposure, dynasore inhibits the cell death pathway triggered by HOS, protecting against ER stress and maintaining a homeostatic level of UPR activity. However, unlike with tBHP exposure, UPR activation due to HOS is independent of PERK and mostly driven by the UPR IRE1 branch. Our results demonstrate the role of the UPR in HOS-driven damage, and the potential of dynasore as a treatment to prevent dry eye epitheliopathy.

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy.

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases.

Relevance of humanized three-dimensional tumor tissue models: a descriptive systematic literature review.

Despite numerous advances in tumor screening, diagnosis, and treatment, to date, tumors remain one of the leading causes of death, principally due to metastasis and the physiological damage produced by tumor growth. Among the main limits related to the study of tumor physiology there is the complex and heterogeneity nature of its environment and the absence of relevant, simple and inexpensive models able to mimic the biological processes occurring in patients allowing the correct clinical translation of results. To enhance the understanding of the mechanisms of tumors and to develop and evaluate new therapeutic approaches the set-up of advanced and alternative models is mandatory. One of the more translational approaches seems to be the use of humanized three-dimensional (3D) tissue culture. This model allows to accurately mimic tumor morphology and biology, maintaining the native microenvironment without any manipulation. However, little is still known on the real clinical relevance of these models for the study of tumor mechanisms and for the screening of new therapy. The aim of this descriptive systematic literature review was to evaluate and summarize the current knowledge on human 3D tumor tissue culture models. We reviewed the strategies employed by researchers to set-up these systems, also considering the different approaches and culture conditions used. All these aspects greatly contribute to the existing knowledge on tumors, providing a specific link to clinical scenarios and making the humanized 3D tumor tissue models a more attractive tool both for researchers and clinicians.

Transcriptome Analysis of Pterygium and Pinguecula Reveals Evidence of Genomic Instability Associated with Chronic Inflammation.

Solar damage due to ultraviolet radiation (UVR) is implicated in the development of two proliferative lesions of the ocular surface: pterygium and pinguecula. Pterygium and pinguecula specimens were collected, along with adjacent healthy conjunctiva specimens. RNA was extracted and sequenced. Pairwise comparisons were made of differentially expressed genes (DEGs). Computational methods were used for analysis. Transcripts from 18,630 genes were identified. Comparison of two subgroups of pterygium specimens uncovered evidence of genomic instability associated with inflammation and the immune response; these changes were also observed in pinguecula, but to a lesser extent. Among the top DEGs were four genes encoding tumor suppressors that were downregulated in pterygium: C10orf90, RARRES1, DMBT1 and SCGB3A1; C10orf90 and RARRES1 were also downregulated in pinguecula. Ingenuity Pathway Analysis overwhelmingly linked DEGs to cancer for both lesions; however, both lesions are clearly still benign, as evidenced by the expression of other genes indicating their well-differentiated and non-invasive character. Pathways for epithelial cell proliferation were identified that distinguish the two lesions, as well as genes encoding specific pathway components. Upregulated DEGs common to both lesions, including KRT9 and TRPV3, provide a further insight into pathophysiology. Our findings suggest that pterygium and pinguecula, while benign lesions, are both on the pathological pathway towards neoplastic transformation.

Deregulated miRNAs in osteoporosis: effects in bone metastasis.

Starting from their role exerted on osteoblast and osteoclast differentiation and activity pathways, microRNAs (miRNAs) have been recently identified as regulators of different processes in bone homeostasis. For this purpose, in a recent review, we highlighted, as deregulated miRNAs could be involved in different bone diseases such as osteoporosis. In addition, recent studies supported the concept that osteoporosis-induced bone alterations might offer a receptive site for cancer cells to form bone metastases, However, to date, no data on specific-shared miRNAs between osteoporosis and bone metastases have been considered and described to clarify the evidence of this link. The main goal of this review is to underline as deregulated miRNAs in osteoporosis may have specific roles in the development of bone metastases. The review showed that several circulating osteoporotic miRNAs could facilitate tumor progression and bone-metastasis formation in several tumor types, i.e., breast cancer, prostate cancer, non-small-cell lung cancer, esophageal squamous cell carcinoma, and multiple myeloma. In detail, serum up-regulation of pro-osteoporotic miRNAs, as well as serum down-regulation of anti-osteoporotic miRNAs are common features of all these tumors and are able to promote bone metastasis. These results are of key importance and could help researcher and clinicians to establish new therapeutic strategies connected with deregulation of circulating miRNAs and able to interfere with pathogenic processes of osteoporosis, tumor progressions, and bone-metastasis formation.

Spinal fusion procedures in the adult and young population: a systematic review on allogenic bone and synthetic grafts when compared to autologous bone.

This systematic review aims to compare clinical evidences related to autologous iliac crest bone graft (ICBG) and non-ICBG (local bone) with allografts and synthetic grafts for spinal fusion procedures in adult and young patients. A systematic search was carried out in three databases (PubMed, Scopus, Web of Science, Cochrane Central Register of Controlled Trials) to identify clinical studies in the last 10 years. The initial search retrieved 1085 studies, of which 24 were recognized eligible for the review. Twelve studies (4 RCTs, 5 prospective, 3 retrospective) were focused on lumbar spine, 9 (2 RCTs, 2 prospective, 4 retrospective, 1 case-series) on cervical spine and 3 (1 RCT, 2 retrospective) on spinal fusion procedures in young patients. Calcium phosphate ceramics, allografts, bioglasses, composites and polymers have been clinically investigated as substitutes of autologous bone in spinal fusion procedures. Of the 24 studies included in this review, only 1 RCT on cervical spine was classified with high level of evidence (Class I) and showed low risk of bias. This RCT demonstrated the safety and efficacy of the proposed treatment, a composite bone substitute, that results in similar and on some metrics superior outcomes compared with local autograft bone. Almost all other studies showed moderately or, more often, high incidence of bias (Class III), thus preventing ultimate conclusion on the hypothesized beneficial effects of allografts and synthetic grafts. This review suggests that users of allografts and synthetic grafting should carefully consider the scientific evidence concerning efficacy and safety of these bone substitutes, in order to select the best option for patient undergoing spinal fusion procedures.

Therapeutic Potential of the Molecular Chaperone and Matrix Metalloproteinase Inhibitor Clusterin for Dry Eye.

Evidence is presented herein supporting the potential of the natural homeostatic glycoprotein CLU (clusterin) as a novel therapeutic for the treatment of dry eye. This idea began with the demonstration that matrix metalloproteinase MMP9 is required for damage to the ocular surface in mouse dry eye. Damage was characterized by degradation of OCLN (occludin), a known substrate of MMP9 and a key component of the paracellular barrier. Following up on this finding, a yeast two-hybrid screen was conducted using MMP9 as the bait to identify other proteins involved. CLU emerged as a strong interacting protein that inhibits the enzymatic activity of MMP9. Previously characterized as a molecular chaperone, CLU is expressed prominently by epithelia at fluid-tissue interfaces and secreted into bodily fluids, where it protects cells and tissues against damaging stress. It was demonstrated that CLU also protects the ocular surface in mouse dry eye when applied topically to replace the natural protein depleted from the dysfunctional tears. CLU is similarly depleted from tears in human dry eye. The most novel and interesting finding was that CLU binds selectively to the damaged ocular surface. In this position, CLU protects against epithelial cell death and barrier proteolysis, and dampens the autoimmune response, while the apical epithelial cell layer is renewed. When present at high enough concentration, CLU also blocks staining by vital dyes used clinically to diagnose dry eye. None of the current therapeutics have this combination of properties to "protect, seal, and heal". Future work will be directed towards human clinical trials to investigate the therapeutic promise of CLU.

Gene therapy for chondral and osteochondral regeneration: is the future now?

Gene therapy might represent a promising strategy for chondral and osteochondral defects repair by balancing the management of temporary joint mechanical incompetence with altered metabolic and inflammatory homeostasis. This review analysed preclinical and clinical studies on gene therapy for the repair of articular cartilage defects performed over the last 10 years, focussing on expression vectors (non-viral and viral), type of genes delivered and gene therapy procedures (direct or indirect). Plasmids (non-viral expression vectors) and adenovirus (viral vectors) were the most employed vectors in preclinical studies. Genes delivered encoded mainly for growth factors, followed by transcription factors, anti-inflammatory cytokines and, less frequently, by cell signalling proteins, matrix proteins and receptors. Direct injection of the expression vector was used less than indirect injection of cells, with or without scaffolds, transduced with genes of interest and then implanted into the lesion site. Clinical trials (phases I, II or III) on safety, biological activity, efficacy, toxicity or bio-distribution employed adenovirus viral vectors to deliver growth factors or anti-inflammatory cytokines, for the treatment of osteoarthritis or degenerative arthritis, and tumour necrosis factor receptor or interferon for the treatment of inflammatory arthritis.

Engineered exosomes: A new promise for the management of musculoskeletal diseases.

BACKGROUND: Exosomes are nanovesicles actively secreted by potentially all cell types, including tumour cells, with the primary role of extracellular systemic communication mediators, both at autocrine and paracrine levels, at short and long distances. Recently, different studies have used exosomes as a delivery system for a plethora of different molecules, such as drugs, microRNAs and proteins. This has been made possible thanks to the simplicity in exosomes engineering, their great stability and versatility for applications in oncology as well as in regenerative medicine. SCOPE OF REVIEW: The aim of this review is to provide information on the state-of-the-art and possible applications of engineered exosomes, both for cargo and specific cell-targeting, in different pathologies related to the musculoskeletal system. MAJOR CONCLUSIONS: The use of exosomes as therapeutic agents is rapidly evolving, different studies explore drug delivery with exosomes using different molecules, showing an enormous potential in various research fields such as oncology and regenerative medicine. GENERAL SIGNIFICANCE: However, despite the significant progress made by the different studies carried out, currently, the use of exosomes is not a therapeutic reality for the considerable difficulties to overcome.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). DESIGN: 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. RESULTS: The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. CONCLUSIONS: NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.

Biomaterials as bone graft substitutes for spine surgery: from preclinical results to clinical study.

Vertebral fusion is performed in order to stabilize the spine in the presence of degenerative, traumatic or oncological pathologies that alter its stability. The autologous bone, harvested from the patient's iliac crest or from the lamina during surgery, is still considered the "gold standard" for spine fusion due to its osteogenic, osteoinductive and osteoconductive properties. However, several biological and synthetic bone substitutes have been introduced as alternatives for regenerating bone tissue. We have studied in particular the use of ceramic biomaterials prepared from hydroxypatite (HA), starting from in vitro analysis, through an in vivo study on ovine animal model and a post-market surveillance analysis, to finally design and perform a clinical study, which is ongoing in our Department. In the first step, HA-derived biomaterials were tested in vitro in the presence of bone marrow-derived human mesenchymal stem cells (hMSCs) and evaluated for their ability to activate precursor cells. In the second step, the biomimetic bone graft substitute SintLife® putty (MgHA) was evaluated in vivo. A posterolateral fusion procedure was applied on 18 sheep, where a fusion level was treated with MgHA, while the other level was treated with autologous bone. Microtomography and histological/histomorphometric analysis were performed six months of after surgery. In the third step, we reported the results of a post-market surveillance study conducted on 4 independent cohorts of patients (total 115 patients), in which HA-derived biomaterials were used as bone graft substitutes or extenders. Finally, a clinical study has been designed and approved by the Ethics Committee of our Institute and is currently ongoing. This study aims to evaluate the efficacy of the ceramic biomaterial SintLife® putty for bone replacement in patients treated by posterolateral fusion for degenerative spine disorders. HA biomaterials were effective in promoting the in vitro growth of hMSCs and their osteogenic differentiation. In the animal model, SintLife® putty has been effective in generating neo-formed bone tissue with morphological and structural features similar to those of the pre-existing bone. The post-market surveillance analysis has not reported any intra-operative nor early or late post-operative adverse events. Seven patients are currently recruited for the clinical trial designed to evaluate Sintlife efficacy for spine fusion (FU range: 1-7 months). No adverse events have been recorded. The first CT analysis performed at 6 months FU showed a good spine fusion. The study is ongoing. Our results, obtained from in vitro, preclinical and clinical studies, suggest that biomaterials derived from hydroxyapatite could be a valid alternative to autologous bone graft for vertebral fusion. This would potentially avoid or reduce the need of autologous bone harvesting and therefore, the risk of drawback-related side effects.

Lipid-lowering effect of bergamot polyphenolic fraction: role of pancreatic cholesterol ester hydrolase.

Bergamot polyphenolic fraction (BPF) has been shown to positively modulate several mechanisms involved in metabolic syndrome, suggesting its use in therapy. In particular, it is able to induce a significant amelioration of serum lipid profile in hyperlipemic patients at different levels. The purpose of our study was to investigate the effect of BPF on cholesterol absorption physiologically mediated by pancreatic cholesterol ester hydrolase (pCEH). An in vitro activity assay was performed to study the effect of BPF on pCEH, whereas the rate of cholesterol absorption was evaluated through in vivo studies. In particular, male, Sprague-Dawley rats (200–225 g) were fed either normal chow or chow supplemented with 0.5% cholic acid, 5.5% peanut oil, and varying amounts of cholesterol (0 to 1.5%). BPF (10 mg/Kg) was daily administrated by means of a gastric gavage to animals fed with lipid supplemented diet for 4 weeks and, at the end of the study, plasma lipids and liver cholesteryl esters were measured in all experimental groups. Our results show that BPF was able to inhibit pCEH activity and this effect was confirmed, in vivo, via detection of lymphatic cholesteryl ester in rats fed with a cholesterol-rich diet. This evidence clarifies a further mechanism responsible for the hypolipemic properties of BPF previously observed in humans, confirming its beneficial effect in the therapy of hypercholesterolemia and in the treatment of metabolic syndrome.

Clusterin in the eye: An old dog with new tricks at the ocular surface.

The multifunctional protein clusterin (CLU) was first described in 1983 as a secreted glycoprotein present in ram rete testis fluid that enhanced aggregation ('clustering') of a variety of cells in vitro. It was also independently discovered in a number of other systems. By the early 1990s, CLU was known under many names and its expression had been demonstrated throughout the body, including in the eye. Its homeostatic activities in proteostasis, cytoprotection, and anti-inflammation have been well documented, however its roles in health and disease are still not well understood. CLU is prominent at fluid-tissue interfaces, and in 1996 it was demonstrated to be the most highly expressed transcript in the human cornea, the protein product being localized to the apical layers of the mucosal epithelia of the cornea and conjunctiva. CLU protein is also present in human tears. Using a preclinical mouse model for desiccating stress that mimics human dry eye disease, the authors recently demonstrated that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration in the tears. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to LGALS3 (galectin-3), a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. CLU depletion from the ocular surface epithelia is seen in a variety of inflammatory conditions in humans and mice that lead to squamous metaplasia and a keratinized epithelium. This suggests that CLU might have a specific role in maintaining mucosal epithelial differentiation, an idea that can now be tested using the mouse model for desiccating stress. Most excitingly, the new findings suggest that CLU could serve as a novel biotherapeutic for dry eye disease.

A cell-based screening assay to identify pharmaceutical compounds that enhance the regenerative quality of corneal repair.

The goal of this study was to develop and validate a simple but quantitative cell-based assay to identify compounds that might be used pharmaceutically to give tissue repair a more regenerative character. The cornea was used as the model, and some specific aspects of repair in this organ were incorporated into assay design. A quantitative cell-based assay was developed based on transcriptional promoter activity of fibrotic marker genes ACT2A and TGFB2. Immortalized corneal stromal cells (HTK) or corneal epithelial cells (HCLE) were tested and compared to primary corneal stromal cells. Cells were transiently transfected with constructs containing the firefly luciferase reporter gene driven by transcriptional promoters for the selected fibrotic marker genes. A selected panel of seven chemical test compounds was used, containing three known fibrosis inhibitors: lovastatin (LOV), tyrphostin AG 1296 (6,7-dimethoxy-3-phenylquinoxaline) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), and four potential fibrosis inhibitors: 5-iodotubercidin (4-amino-5-iodo-7-(ß-D-ribofuranosyl)-pyrrolo(2,3-d)pyrimidine), anisomycin, DRB (5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole) and latrunculin B. Transfected cells were treated with TGFB2 in the presence or absence of one of the test compounds. To validate the assay, compounds were tested for their direct effects on gene expression in the immortalized cell lines and primary human corneal keratocytes using RT-PCR and immunohistochemistry. Three "hits" were validated LOV, SB203580 and anisomycin. This assay, which can be applied in a high throughput format to screen large libraries of uncharacterized compounds, or known compounds that might be repurposed, offers a valuable tool for identifying new treatments to address a major unmet medical need. Anisomycin has not previously been characterized as antifibrotic, thus, this is a novel finding of the study.

Past and present of interposition arthroplasties for joint repair with special tribute to the contribution by Vittorio Putti.

Several techniques have been proposed to restore the compromised function of a joint. These include the arthroplasty by placing various tissues or materials between the articular surfaces. An important contribution to the diffusion of arthroplasty techniques was made by Vittorio Putti, head of the Rizzoli Orthopedic Institute in Bologna from 1912 to 1940. Interposition arthroplasty is still used for some non-weight-bearing joints, such as wrist and elbow, and gives good results. This type of surgery has been further developed by the improvement in biomaterials, biomechanical studies and the regenerative medicine. This paper describes the development starting from a historical survey particularly focused on Putti's contribution and ending with the state of the art of regenerative medicine in the treatment of joint diseases. Level of evidence V.

Glaucomatous MYOC mutations activate the IL-1/NF-κB inflammatory stress response and the glaucoma marker SELE in trabecular meshwork cells.

PURPOSE: Activation of the IL-1/NF-κB inflammatory stress pathway and induction of SELE expression in the trabecular meshwork (TBM) is a marker for high-tension glaucomas of diverse etiology. Pathway activation stimulates aqueous outflow and protects against oxidative stress, but may be damaging in the long-term. MYOC mutations have been causally linked to high-tension forms of primary open angle glaucoma (POAG). This study investigated a possible link between MYOC mutations and activation of the IL-1/NF-κB pathway and expression of SELE. METHODS: We constructed MYOC expression vectors with mutations at sites that cause POAG. Mutations (Q368X, Y437H, A427T) were selected to represent proteins with differing POAG-causing potency (Q368X > Y437H > A427T) and intracellular retention behavior (Q368X and Y437H retained, A427T released). The constructs were made in two different kinds of vectors; one a plasmid designed for transient transfection (pCMV6), and one a doxycycline-inducible lentiviral vector (pSLIK) for stable cell transduction. The immortalized human trabecular meshwork line TM-1 was used for all expression studies. Expression of IL1A mRNA was determined by reverse transcription (RT)-PCR, as well as a set of five other genes associated with signaling pathways linked to glaucoma: IL1B and IL6 (NF-κB pathway), TGFB2 and ACTA2 (TGF-ß pathway) and FOXO1 (E2F1 apoptotic pathway). An ELISA was used to quantify IL1A protein released into culture media. To quantify intracellular NF-κB activity, we transiently transfected stably transduced cell lines with a luciferase expression vector under control of the IL8 promoter (containing an NF-κB response element). RESULTS: Transiently expressed wild-type MYOC was released into cell culture media, whereas mutant MYOCs Q368X and Y437H remained within cells. Both mutant MYOCs activated the IL-1/ NF-κB pathway, significantly stimulating expression of IL1A and IL1B. However Y437H, which causes a severe glaucoma phenotype, was less effective than Q368X, which causes a moderate glaucoma phenotype. In addition, the retained mutants stimulated expression of stress response genes ACTA2 and FOXO1. Unexpectedly, wild-type MYOC significantly decreased expression of IL6 and TGFB2, to approximately half of the control levels, and expression of IL1B and ACTA2 was also slightly decreased. Induction of MYOC mutants Q368X and Y437H in stably transduced cell lines significantly stimulated the level of IL1A protein released into culture media. Once again however, the effect of the severe MYOC mutant Y437H was less than the effect of the moderate MYOC mutant Q368X. In contrast, induced expression of the intracellularly retained mutant MYOC A427T or wild-type MYOC did not change the amount of IL1A protein in culture media. Induction of Y437H MYOC plus IL1A treatment increased NF-κB activity by 25% over IL1A alone. In contrast, induction of Q368X or A427T plus IL1A treatment did not significantly affect NF-κB activity over IL1A alone. However, wild-type MYOC expression inhibited IL1A-stimulated NF-κB activity. We also observed that endogenous MYOC expression was induced by IL1A in TM-1 cells and primary TBM cell cultures. SELE was co-expressed with MYOC in the primary cell lines. CONCLUSIONS: These results indicate that POAG-causing MYOC mutants activate the IL-1/NF-κB pathway, with activation levels correlated with intracellular retention of the protein, but not POAG-causing potency. Unexpectedly, it was also discovered that wild-type MYOC inhibits activation of the IL-1/NF-κB pathway, and that activation of the IL-1/NF-κB pathway stimulates expression of MYOC. This is the first evidence that glaucoma-causing MYOC mutants can activate the inflammatory response and that wild-type MYOC has anti-inflammatory activity.

Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice.

PURPOSE: Previously, we demonstrated that scleral stem/progenitor cells (SSPCs) from mice have a chondrogenic differentiation potential, which is stimulated by transforming growth factor-ß (TGF-ß). In the present study, we hypothesized that chondrogenesis in the sclera could be a possible mechanism in myopia development. Therefore, we investigated the association of form-deprivation myopia (FDM) with expressions in mice sclera representing the chondrogenic phenotype: collagen type II (Col2) and α-smooth muscle actin (α-SMA). METHODS: The mRNA levels of α-SMA and Col2 in cultured murine SSPCs during chondrogenesis stimulated by TGF-ß2 were determined by real-time quantitative RT-PCR (qRT-PCR). The expression patterns of α-SMA and Col2 were assessed by immunohistochemistry in a three dimensional pellet culture. In an FDM mouse model, a western blot analysis and immunofluorescence study were used to detect the changes in the α-SMA and Col2 protein expressions in the sclera. In the RPE-choroid complex, qRT-PCR was used to detect any changes in the TGF-ß mRNA expression. RESULTS: The treatment of SSPCs in vitro with TGF-ß2 for 24 h at 1 or 10 ng/ml led to increased levels of both the α-SMA and Col2 expressions. In addition, we observed the formation of cartilage-like pellets from TGF-ß2-treated SSPCs. Both α-SMA and Col2 were expressed in the pellet. In an in-vivo study, the α-SMA and Col2 protein expressions were significantly increased in the sclera of FDM eyes in comparison to contralateral control eyes. Similarly, the levels of TGF-ß in the RPE-choroid complex of an FDM eye were also significantly elevated. CONCLUSION: Based on the concept of stem cells possessing multipotent differentiation potentials, scleral chondrogenesis induced by SSPCs may play a role in myopia development. The increased expressions of the cartilage-associated proteins Col2 and α-SMA during scleral chondrogenesis may be potential markers for myopia development. In addition, the increased levels of TGF-ß mRNA in the RPE-choroid complex might induce the chondrogenic change in the sclera during myopia development.

Metabolic and cytoprotective effects of in vivo peri-patellar hyaluronic acid injections in cultured tenocytes.

The purpose of this study was to investigate tenocyte mechanobiology after sudden-detraining and to examine the hypothesis that repeated peri-patellar injections of hyaluronic acid (HA) on detrained patellar tendon (PT) may reduce and limit detrained-associated damage in tenocytes. Twenty-four male Sprague-Dawley rats were divided into three groups: Untrained, Trained and Detrained. In the Detrained rats, the left tendon was untreated while the right tendon received repeated peri-patellar injections of either HA or saline (NaCl). Tenocyte morphology, metabolism and synthesis of C-terminal-propeptide of type I collagen, collagen-III, fibronectin, aggrecan, tenascin-c, interleukin-1ß, matrix-metalloproteinase-1 and-3 were evaluated after 1, 3, 7 and 10 days of culture. Transmission-electronic-microscopy showed a significant increase in mitochondria and rough endoplasmic reticulum in cultured tenocytes from Detrained-HA with respect to those from Detrained-NaCl. Additionally, Detrained-HA cultures showed a significantly higher proliferation rate and viability, and increased synthesis of C-terminal-Propeptide of type I collagen, fibronectin, aggrecan, tenascin-c and matrix-metalloproteinase-3 with respect to Detrained-NaCl ones, whereas synthesis of matrix-metalloproteinase-1 and interleukin-1ß was decreased. Our study demonstrates that discontinuing training activity in the short-term alters tenocyte synthetic and metabolic activity and that repeated peri-patellar infiltrations of HA during detraining allow the maintenance of tenocyte anabolic activity.