Intravitreal Pharmacokinetic Study of the Antiangiogenic Glycoprotein Opticin.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.

Fibromodulin reduces scar size and increases scar tensile strength in normal and excessive-mechanical-loading porcine cutaneous wounds.

Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive-mechanical-loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule-fibromodulin (FMOD) protein-can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive-mechanical-loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD-based technologies hold high translational potential and applicability to human patients suffering from scarring-especially hypertrophic scarring.

Nanofiber-based hydrogels with extracellular matrix-based synthetic peptides for the prevention of capsular opacification.

Nanofiber-based hydrogels (nanogels) with different, covalently bound peptides were used as an extracellular environment for lens epithelial cells (LECs) in order to modulate the capsular opacification (CO) response after lens surgery in a porcine eye model. Lenses were divided into 15 groups (n = 4 per group), the lens content was removed and the empty capsules were refilled with nanogel without peptides and nanogels with 13 combinations of 5 different peptides: two laminin-derived, two fibronectin-derived, and one collagen IV-derived peptide representing cell adhesion motifs. A control group of 4 lenses was refilled with hyaluronan. After refilling, lenses were extracted from the porcine eye and cultured for three weeks. LECs were assessed for morphology and alpha smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. Compared to hyaluronan controls, lenses filled with nanogel had less CO formation, indicated by a lower αSMA expression (P = 0.004). Microscopy showed differences in morphological cell response within the nanogel refilled groups. αSMA expression in these groups was highest in lenses refilled with nanogel without peptides (9.54 ± 11.29%). Overall, LEC transformation is reduced by the presence of nanogels and the response is improved even further by incorporation of extracellular matrix peptides representing adhesion motifs. Thus, nanomaterials targeting biological pathways, in our case interactions with integrin signaling, are a promising avenue toward reduction of CO. Further research is needed to optimize nanogel-peptide combinations that fully prevent CO.

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors.

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.

RHAMM (receptor hyaluronan-mediated motility)-target peptides induce apoptosis in prostate cancer cells.

In this work the effect of RHAMM (receptor hyaluronan-mediated motility)-target peptides was investigated on the viability, apoptosis and necrosis of prostate cancer cells (PC3m-LN4). It has been established that RHAMM-target peptides inhibited on 90 % cell viability of PC3m-LN4 cells at a concentration of 10 ug / ml (2х10-7 M) for 48 h. It has shown that RHAMM-target peptides induced apoptosis and inhibited necrosis of tumor cells. RHAMM-target peptide had no effect on fibroblasts (non-tumor cells) and fibroblasts (RHAMM-/-). The studies also revealed that RHAMM-target peptides enhanced activity of caspase-3/7 in cancer cells.

Reelin supplementation recovers synaptic plasticity and cognitive deficits in a mouse model for Angelman syndrome.

The Reelin signaling pathway is implicated in processes controlling synaptic plasticity and hippocampus-dependent learning and memory. A single direct in vivo application of Reelin enhances long-term potentiation, increases dendritic spine density and improves associative and spatial learning and memory. Angelman syndrome (AS) is a neurological disorder that presents with an overall defect in synaptic function, including decreased long-term potentiation, reduced dendritic spine density, and deficits in learning and memory, making it an attractive model in which to examine the ability of Reelin to recover synaptic function and cognitive deficits. In this study, we investigated the effects of Reelin administration on synaptic plasticity and cognitive function in a mouse model of AS and demonstrated that bilateral, intraventricular injections of Reelin recover synaptic function and corresponding hippocampus-dependent associative and spatial learning and memory. Additionally, we describe alteration of the Reelin profile in tissue from both the AS mouse and post-mortem human brain.

Targeted delivery of extracellular matrix protected against neurologic defects after focal ischemia reperfusion in rats.

Ischemic stroke is one of the leading causes of morbidity and mortality worldwide and characterized by defective angiogenesis. The functional sequences (RGDs, GRGDSPASSPISC) derived from fibronectin have been confirmed to augment angiogenesis in vivo and in vitro. However, delivery of peptides into the brain parenchyma has been hampered by the presence of the blood-brain barrier (BBB). We fused RGDs with penetratin (Antp) derived from Drosophila antennapedia homeodomain protein to improve the penetration of peptides through BBB into ischemic hemisphere. We found Antp-RGDs successfully not only penetrate the SH-SY5Y cells but also penetrated through BBB into ischemic hemisphere by intraperitoneal injection. In addition, application of Antp-RGDs to the focal cerebral ischemic reperfusion injury in rats resulted in the reduction of cerebral ischemic volume and the improvement of neurologic score according to the 21-point score. We further demonstrated that activation of phosphorylation-extracellular-signal related kinase 1/2 (p-ERK 1/2) and upregulation of gene VEGF resulted from post-treatment with Antp-RGDs 2 hours after reperfusion onset might at least partly contribute to the benefic changes after focal cerebral ischemic reperfusion injury in rats. Our data suggested that Antp-RGDs may serve as an attractive therapeutic intervention for treating ischemic stroke.

The extracellular matrix protein mindin as a novel adjuvant elicits stronger immune responses for rBAG1, rSRS4 and rSRS9 antigens of Toxoplasma gondii in BALB/c mice.

BACKGROUND: Vaccines are the most effective agents to control infections. However, recombinant vaccines often do not elicit strong immune responses. Protein antigens combined with proper adjuvants have been widely used to induce immune responses, especially the humoral immune responses, against various pathogens, including parasites. The extracellular matrix protein mindin has been recognised as an immune facilitator for initiating innate immune responses. It has therefore been expected to be a potentially potent adjuvant in the development of novel vaccines. The aim of this study was to investigate whether mindin could facilitate the induction of antigen-specific immune responses to recombinant antigens (rBAG1, rSRS4 and rSRS9) of Toxoplasma gondii in BALB/c mice. METHODS: In this study, we explored the adjuvant effect of the recombinant mindin in the generation of specific Th1 and Th2 responses to each of three T. gondii antigens, BAG1, SRS4 and SRS9. All mice in the experimental groups received either antigen alone or in combination with Freund's adjuvant or with the recombinant mindin. The immune responses after immunisation were measured by ELISA and lymphoproliferative assays. The immunised mice were challenged with live T. gondii tachyzoites, and the protection efficiency was compared between the groups. RESULTS: Our results revealed that mindin as an adjuvant could facilitate the recombinant proteins to efficiently stimulate humoral and cellular responses, including antigen-specific IgG1 and IgG2a, as well as lymphocyte proliferation. Furthermore, significantly improved protection against T. gondii infection was observed in the mindin group compared with that of Freund's adjuvant and no-adjuvant groups. CONCLUSIONS: The extracellular matrix protein mindin can effectively induce antigen-specific humoral and cell-mediated immune responses. Our study provides a valuable basis for the development of an efficient, safe, non-toxic vaccine adjuvant for future use in humans and animals.

Effects of ECM protein mimetics on adhesion and proliferation of chorion derived mesenchymal stem cells.

BACKGROUND: We evaluated the effects of fibronectin, collagen, cadherin, and laminin based extracellular matrix (ECM) protein mimetics coated with mussel derived adhesive protein (MAP) on adhesion and proliferation of chorionic mesenchymal stem cells (cMSCs). METHODS: Human placental chorionic tissues from term third-trimester pregnancies (n=3) were used. The cMSCs were cultured on rationally designed ECM protein mimetics coated with MAP on plastic surfaces with the addition of reduced fetal bovine serum (0.5%, 1% FBS). Adhesion capabilities were monitored by a real time cell analysis system (RTCA) utilizing an impedance method. Proliferation capabilities were monitored by RTCA and MTS assay. RESULTS: Of the ECM protein mimetics tested, GRGDSP(FN) coated surfaces exhibited the highest adhesion and proliferation capabilities on RTCA at FBS concentration of 0.5% and 1%. When 0.5% FBS was added to ECM protein mimetics during the MTS assay, GRGDSP(FN), REDV(FN), and collagen mimetics, GPKGAAGEPGKP(ColI) showed higher cMSCs proliferation compared with the control. When 1% FBS was added, GRGDSP(FN) and TAIPSCPEGTVPLYS(ColIV) showed significant cMSCs proliferation capacity. CONCLUSIONS: Fibronectin mimetics, GRGDSP(FN) amino acid sequence showed the highest adhesion and proliferation capabilities. In addition, results from RTCA assessment of cell viability correlated well with the tetrazolium-based MTS assay.

Effect of amelogenin ECM protein on the healing of chronic leg ulcers with atrophie blanche.

OBJECTIVE: To determine the effect of topically applied amelogenin extracellular matrix protein(AEMP) in patients with non-healing venous leg ulcers combined with atrophie blanche. METHOD: This retrospective case series of patients with non-healing venous leg ulcers with atrophie blanche of the distal proportion of their lower legs, where non-healing was defined as no progress toward healing for 3 months previously, under standard therapy. Patient records were reviewed for associated diseases, wound diagnoses, distal blood pressure, previous treatments and changes in wound area. Patients were treated with AEMP once a week, for a period of 12 weeks, or until full healing. RESULTS: Eleven patient records were reviewed retrospectively. The median age of the patients was 81 years (range 40-95 years), with a mean wound size of 4.7 ± 3.Scm2 and median wound duration of 6 months (range 3-444 months).AII patients had venous or combined arterial/venous insufficiency. After 12 weeks' treatment with AEMP, complete healing, defined as I 00% re-epithelialisation, was documented in four patients (36%), marked improvement(> SO% epithelialisation) in three patients (54%, 55% and 83% wound closure, respectively), slight improvement in one patient (9.4% wound closure), no change for two patients and worsening in one.AEMP was well tolerated, and no patients reported side effects. CONCLUSION: The results of this retrospective study suggest that AEMP improves healing in chronic venous leg ulcers combined with atrophie blanche.

High-dose RHAMM-R3 peptide vaccination for patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma.

BACKGROUND: Recently, we demonstrated immunological and clinical responses to a RHAMM-R3 peptide vaccine in patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma. To improve the outcome of the vaccine, a second cohort was vaccinated with a higher dose of 1,000 microg peptide. DESIGN AND METHODS: Nine patients received four vaccinations subcutaneously at a biweekly interval. Immunomonitoring of cytotoxic CD8(+) as well as regulatory CD4(+) T cells was performed by flow cytometry as well as by enzyme-linked immunospot (ELISpot) assays. Parameters of clinical response were assessed. RESULTS: In 4 of 9 patients (44%) we detected positive immunological responses. These patients showed an increase of CD8(+)RHAMM-R3_tetramer(+)/CD45RA(+)/CCR7(-)/CD27(-)/CD28(-) effector T cells and an increase of R3-specific CD8+ T cells. Two of these patients showed a significant decrease of regulatory T cells (Tregs). In one patient without response Tregs frequency increased from 5 to 16%. Three patients showed clinical effects: one patient with myelodysplastic syndrome RAEB-1 showed a reduction of leukemic blasts in the bone marrow, another myelodysplastic syndrome patient an improvement of peripheral blood counts and one patient with multiple myeloma a reduction of free light chains. Clinical and immunological reactions were lower in this cohort than in the 300 microg cohort. CONCLUSIONS: High-dose RHAMM-R3 peptide vaccination induced immunological responses and positive clinical effects. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematologic malignancies. However, higher doses of peptide did not improve the frequency and intensity of immune responses in this trial.

Recombinant decorin ameliorates the pulmonary structure alterations by down-regulating transforming growth factor-beta1/SMADS signaling in the diabetic rats.

This study investigated the role of transforming growth factor-beta1 (TGF-beta1)/Smads signaling in the alterations of lung structure and the effect of the exogenous decorin on lung structure modification in streptozotocin (STZ)-induced diabetic rats. Seventy-two Sprague-Dawley rats were evenly divided into four groups: STZ-induced diabetic rats (diabetic control), decorin adenovirus vector (Ad)-treated STZ rats (Ad-DCN), Ad-lacZ-treated STZ rats (Ad-lacZ), and normal controls. At 8, 16, and 28 weeks after STZ treatment, haematoxylin-eosin (H&E) and Masson's trichrome staining were performed to investigate the histological changes of diabetic lungs; Expressions of TGF-beta1 and collagen type IV in the diabetic lung were measured by Western blot and immunohistochemistry. Phosphorylated Smad2 (P-Smad2), one of the major TGF-beta1 receptor substrates, was also detected using Western blot. The histological changes of diabetic lung included obvious inflammatory cell infiltration and moderate expanding of alveolar septum stained as collagen. Immunolabeled collagen type IV increased in the alveolar septa in the diabetic lung. Activities of TGF-beta1/Smads signaling increased in the diabetic lung during the 28 weeks of diabetes (p < 0.05 vs. control), and positive staining of TGF-beta1 was mainly found in the cytoplasm of the infiltrated inflammatory cells. Exogenous decorin effectively suppressed the increased activities of TGF-beta1/Smads signaling and partly attenuated collagen deposits in the alveolar septum. Increased activity of TGF-beta1/Smads signaling might play a critical role in the accumulation of collagen in the diabetic lung. The protective effect of decorin in the diabetic lung is at least partly because of the down-regulation of the TGF-beta1/Smads signaling.

Amelogenin (an extracellular matrix protein) application on ischemic colon anastomosis in rats.

BACKGROUND: Ischemia is a troublesome problem that can cause intestinal emergencies and complicate the treatment. Identification of a chemical agent with beneficial effects on the healing process in risky colon anastomosis with the aim of reducing leakage rates is a popular topic in the era of surgical research. Data is lacking about the role of amelogenin, an extracellular matrix protein, during the healing process of gastrointestinal anastomosis. In this study, the effects of amelogenin treatment on ischemic colon anastomosis were evaluated. METHODS: Adult male Wistar Albino rats weighing 200-250 g were divided into three weight-matched groups as normal colon anastomosis group (n=8), ischemic colon anastomosis group (n=8), and amelogenin-treated ischemic colon anastomosis group (n=8). Sufficient equal volume of amelogenin to cover the anastomosis area entirely was applied topically. All animals were sacrificed on postoperative day four. Bursting pressure levels were measured. Peri-anastomotic colon tissue hydroxyproline levels were also assessed. RESULTS: Bursting pressure level of the ischemic colon anastomosis group was significantly lower than the normal colon anastomosis and the amelogenin-treated ischemic colon anastomosis groups, respectively (p=0.006, p=0.008). CONCLUSION: Amelogenin treatment supports the physical strength of ischemic colon anastomosis.

Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

Triple-negative breast cancer (TNBC) patients have the worst prognosis and distant metastasis-free survival among all major subtypes of breast cancer. The poor clinical outlook is further exacerbated by a lack of effective targeted therapies for TNBC. Here we show that ectopic expression and therapeutic delivery of the secreted protein Tubulointerstitial nephritis antigen-like 1 (Tinagl1) suppresses TNBC progression and metastasis through direct binding to integrin α5ß1, αvß1, and epidermal growth factor receptor (EGFR), and subsequent simultaneous inhibition of focal adhesion kinase (FAK) and EGFR signaling pathways. Moreover, Tinagl1 protein level is associated with good prognosis and reversely correlates with FAK and EGFR activation status in TNBC. Our results suggest Tinagl1 as a candidate therapeutic agent for TNBC by dual inhibition of integrin/FAK and EGFR signaling pathways.

Reduction in facial photodamage by a topical growth factor product.

A topical gel containing a proprietary mixture of over 110 growth factors, cytokines, and soluble matrix proteins secreted by human dermal fibroblasts was evaluated for safety and efficacy in the treatment of mild to severe facial photodamage. In a double-blind study, 60 subjects were randomly assigned to receive either active gel or the vehicle and applied twice daily for 6 months along with a moisturizing cleanser and sunscreen. Efficacy (profilometry, photography, and clinical assessment) and safety (adverse event reporting) measures were evaluated at 0, 3, and 6 months. Treatment with the active gel for 3 months produced greater reduction in fine lines and wrinkles than the vehicle treatment as measured by objective and subjective assessment techniques. The results were either statistically significant (P < or = .05) or trending towards statistical significance (P < or = .1). This study demonstrates that addition of a topical formulation of growth factors and cytokines to a basic skin care regimen reduces the signs of photoaging.

Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice.

BACKGROUND: Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. METHODS: A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). RESULTS: The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. CONCLUSION: Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.

Short Link N promotes disc repair in a rabbit model of disc degeneration.

BACKGROUND: The degeneration of the intervertebral disc (IVD) is characterized by proteolytic degradation of the extracellular matrix, and its repair requires the production of an extracellular matrix with a high proteoglycan-to-collagen ratio characteristic of a nucleus pulposus (NP)-like phenotype in vivo. At the moment, there is no medical treatment to reverse or even retard disc degeneration. The purpose of the present study was to determine if a low dose of short link N (sLN), a recently discovered fragment of the link N peptide, could behave in a manner similar to that of link N in restoring the proteoglycan content and proteoglycan-to-collagen ratio of the disc in a rabbit model of IVD degeneration, as an indication of its potential therapeutic benefit in reversing disc degeneration. METHODS: Adolescent New Zealand white rabbits received an annular puncture with an 18-gauge needle into two noncontiguous discs to induce disc degeneration. Two weeks later, either saline (10 µL) or sLN (25 µg in 10 µL saline) was injected into the center of the NP. The sLN concentration was empirically chosen at a lower molar concentration equivalent to half that of link N (100 µg in 10 µL). The effect on radiographic, biochemical and histologic changes were evaluated. RESULTS: Following needle puncture, disc height decreased by about 25-30% within 2 weeks and maintained this loss for the duration of the 12-week study; a single 25-µg sLN injection at 2 weeks partially restored this loss in disc height. sLN injection led to an increase in glycosaminoglycans (GAG) content 12 weeks post-injection in both the NP and annulus fibrosus (AF). There was a trend towards maintaining control disc collagen-content with sLN supplementation and the GAG-to-collagen ratio in the NP was increased when compared to the saline group. CONCLUSIONS: When administered to the degenerative disc in vivo, sLN injection leads to an increase in proteoglycan content and a trend towards maintaining control disc collagen content in both the NP and AF. This is similar to link N when it is administered to the degenerative disc. Thus, pharmacologically, sLN supplementation could be a novel therapeutic approach for treating disc degeneration.

The effect of molecules in mother-of-pearl on the decrease in bone resorption through the inhibition of osteoclast cathepsin K.

This study evaluates the effect of the mother-of-pearl (nacre) organic matrix on mammalian osteoclast activity and on cathepsin K protease. Rabbit osteoclasts were cultured on bovine cortical bone slices in the presence of water-soluble molecules extracted from nacre of the pearl oyster Pinctada margaritifera. Osteoclast resorption activity was determined by quantification of the resorption surface area on bovine bone slices. Papain and cathepsin K, B and L inhibition tests were performed in the presence of the nacre water-soluble extracts. The active crude extract was fractionated by dialysis and reversed-phase high-performance liquid chromatography before electrospray mass spectrometry analysis of inhibitory fractions. The water-soluble molecules extracted from nacre decreased bone resorption without jeopardizing osteoclast survival. The hydrolytic activity of cysteine proteinases was reduced when the enzymes were incubated with the nacre water-soluble molecules. Trending towards characterization of the molecules involved, it appears that cathepsin K inhibitors remain in different nacre water-soluble organic matrix subfractions, composed of low molecular weight molecules. Mollusk shell nacre contains molecules capable of reducing osteoclast bone resorption activity by inhibiting cathepsin K, giving a new facet of the bioactivity of nacre as bone biomaterial.

ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth.

Tissue engineering using a combination of biomaterials and cells represents a new approach to nerve repair. We have investigated the effect that extracellular matrix (ECM) molecules have on Schwann cell (SC) attachment and proliferation on the nerve conduit material poly-3-hydroxybutyrate (PHB), and SC influence on neurite outgrowth in vitro. Initial SC attachment to PHB mats was unaffected by ECM molecules but proliferation increased (laminin > fibronectin > collagen). SCs seeded onto ECM-coated culture inserts suspended above a monolayer of NG108-15 cells determined the effect of released diffusible factors. The effect of direct contact between the two cell types on ECM molecules was also investigated. In both systems SCs enhanced neurite number per cell and percentage of NG108-15 cells sprouting neurites. NG108-15 cells grown in direct contact with SCs had significantly longer neurites than those exposed to diffusible factors when seeded on laminin or fibronectin. Diffusible factors released from SCs cultured on ECM molecules appear to initiate neurite outgrowth, whereas SC-neuron contact promotes neurite elongation. SC proliferation was maximal on poly-D-lysine-coated surfaces, but these cells did not influence neurite outgrowth to the levels of laminin or fibronectin. This suggests that ECM molecules enhance cell number and activate SCs to release neurite promoting factors. Addition of ECM molecules to PHB nerve conduits containing SCs is likely to provide benefits for the treatment of nerve injuries.

Arthritis induced with minor cartilage proteins.

Type XI collagen (CXI) and cartilage oligomeric matrix protein (COMP) are minor components in cartilage, shown to be arthritogenic. CXI is a heterotrimeric triple helical fibrillar collagen and intermingled in the collagen fibers with type II (CII). COMP is the major noncollagenous protein of cartilage and is a homopentamer, interacting with the collagen fibers with each of its subunits. Similar to CII, homologous rat CXI also induces a chronic arthritis in rats but with a different major histocompatibility complex (MHC) genetic control and pathogenesis. CXI induced arthritis (C(XI)IA) is characterized by a more pronounced chronic relapsing disease course. The MHC allele of importance is the RT1f haplotype and, surprisingly, some of the CII associated MHC alleles like RT1a are less permissive. Immunization with COMP induces a severe but self-limited arthritis in strains with a genetic background resistant to most other forms of arthritis or even autoimmune models, the E3 rat. The MHC association also differs between the different models (CIA, CXI, and COMPIA). An autoimmune response to COMP is triggered despite the circulation of COMP fragments in both physiologic and arthritic states. The induction of arthritis in rats with CXI or COMP provides an arthritis models with a distinct pathogenesis as compared with other induced arthritis models.