Forward Light Scattering of the Vitreous Gel After Enzymatic Aging: An In Vitro Model to Study Vitreous Opacification.

Purpose: Symptomatic vitreous opacifications, so-called floaters, are difficult to objectively assess majorly limiting the possibility of in vitro studies. Forward light scattering was found previously to be increased in eyes with symptomatic floaters. Using an objective setup to measure forward light scattering, we studied the effects of enzymatically digesting the components of the vitreous body on straylight to develop an in vitro model of vitreous opacifications. Methods: Fifty-seven porcine vitreous bodies were digested using hyaluronidase, collagenase, trypsin, and bromelain, as well as using a combination of hyaluronidase + collagenase and hyaluronidase + bromelain. A modified C-Quant setup was used to objectively assess forward light scattering. Results: Depletion of hyaluronic acid majorly increased vitreous straylight (mean increase 34.4 deg2/sr; P = 0.01), whereas primarily digesting the vitreous gel with collagenase or trypsin did not significantly affect straylight. When collagenase or bromelain is applied in hyaluronic acid depleted vitreous gels, the increase in forward light scattering is reversed partially. Conclusions: The age-related loss of hyaluronic acid primarily drives the increase in vitreous gel straylight induced by conglomerates of collagen. This process can be reversed partially by digesting collagen. This in vitro model allows the objective quantification and statistical comparison of straylight burden caused by vitreous opacities and, thus, can serve as a first testing ground for pharmacological therapies, as demonstrated with bromelain.

Development on potential skin anti-aging agents of Cosmos caudatus Kunth via inhibition of collagenase, MMP-1 and MMP-3 activities.

BACKGROUND: Skin aging is associated with degradation of collagen by matrix metalloproteinases (MMPs), which leads to loss of skin elasticity and formation of wrinkles. Cosmos caudatus Kunth (CC) has been traditionally claimed as an anti-aging agent in Malaysia. Despite its well-known antioxidant activity, the anti-aging properties of CC was not validated. PURPOSE: This study aimed to investigate the anti-aging potential of CC extracts and fractions, particularly their inhibition of collagenase, MMP-1 and MMP-3 activities in human dermal fibroblasts CCD-966SK, followed by isolation, identification and analysis of their bioactive constituents. STUDY DESIGN AND METHODS: DPPH assay was firstly used to evaluate the antioxidant activity throughout the bioactivity-guided fractionation. Cell viability was determined using MTS assay. Collagenase activity was examined, while MMP-1 and MMP-3 expression were measured using qRT-PCR and western blotting. Then, chemical identification of pure compounds isolated from CC fractions was done by using ESIMS, 1H and 13C NMR spectroscopies. HPLC analyses were carried out for bioactive fractions to quantify the major components. RESULTS: Throughout the antioxidant activity-guided fractionation, fractions CC-E2 and CC-E3 with antioxidant activity and no toxicity towards CCD-966SK cells were obtained from CC 75% ethanol partitioned layer (CC-E). Both fractions inhibited collagenase activity, MMP-1 and MMP-3 mRNA and protein expression, as well as NF-κB activation induced by TNF-α in CCD-966SK cells. 14 compounds, which mainly consists of flavonoids and their glycosides, were isolated. Quercitrin (14.79% w/w) and quercetin (11.20% w/w) were major compounds in CC-E2 and CC-E3, respectively, as quantified by HPLC. Interestingly, both fractions also inhibited the MMP-3 protein expression synergistically, compared with treatment alone. CONCLUSION: The quantified CC fractions rich in flavonoid glycosides exhibited skin anti-aging effects via the inhibition of collagenase, MMP-1 and MMP-3 activities, probably through NF-κB pathway. This is the first study reported on MMP-1 and MMP-3 inhibitory activity of CC with its chemical profile, which revealed its potential to be developed as anti-aging products in the future.

A Comparison of Topical Agents for Eschar Removal in a Porcine Model: Bromelain-enriched vs Traditional Collagenase Agents.

Surgical excision and grafting of deep partial-thickness (DPT) and full-thickness (FT) burns is a cornerstone of wound care. The use of commercially available topical enzymatic agents has been limited due to slower and less complete eschar removal than surgical excision. Using a porcine model of DPT and FT burns, we compared the eschar removal efficacy of a bromelain-enriched enzymatic agent derived from the stems of pineapple plants and a commercially available collagenase. We created 40 DPT and 40 FT burns on four anesthetized Yorkshire pigs. Eschar removal was initiated 24 hours later. Two pigs each were randomly assigned to collagenase or the bromelain-enriched agent. The bromelain-enriched agent was applied topically once for 4 hours followed by a 2-hour soaking. The collagenase was applied topically daily until complete removal of eschar or for up to 14 days. All bromelain-enriched treated FT burns underwent complete removal of the eschar after a single application while none of the collagenase-treated FT burns underwent complete removal of the eschar even after 14 days of treatment. All bromelain-enriched treated DPT burns had complete eschar removal after the single application. None of the collagenase-treated DPT burns experienced complete removal of eschar after 10 days; by day 14, 35% had complete eschar removal, 30% had >50% eschar removed, and 35% had <50% eschar removed. We conclude that eschar removal is quicker and more complete with the bromelain-enriched compared with collagenase debriding agent.

Novel strategy of cartilage repairing via application of P. atlantica with stem cells and collagen.

Osteoarthritis (OA) is an inflammatory joint condition, still lacking effective treatments. Some factors consider as the main causes of OA, including biochemical, mechanical, and genetic factors. The growth of studies confirmed that modern medicine in combination with folk medicine regarding the arrival of reliable, efficient, and safe therapeutic products against OA. In the present study, the effects of various single and combinatorial treatments of knee articular cartilage, including stem cells, collagen, and P. atlantica hydroalcoholic leaves extract were investigated in a rat-induced OA model. On week 12 after OA confirmation, histopathology and radiography assessments were evaluated and the serum and synovial fluid levels of TAC, TNF-α, PEG2, MPO, MMP3, MMP13, and MDA were also measured. Combination therapy of OA-induced rats with hydroalcoholic extract of P. atlantic leaves, stem cells, and collagen considerably increased the efficacy of treatment as evidenced by increasing the TAC and lowering TNF-α, MPO, MMP3, and MMP13 compared to control group and even groups received single therapy. This is in agreement with a high amount of total phenolic compounds and antioxidant capacities of the hydroalcoholic extract of P. atlantic leaves. It is concluded that multifunctional agents targeting the pathophysiology of OA has exhibited significant therapeutic effects against OA.

Mild Acid-Responsive "Nanoenzyme Capsule" Remodeling of the Tumor Microenvironment to Increase Tumor Penetration.

Dense extracellular matrix (ECM) severely impedes the spread of drugs in solid tumors and induces hypoxia, reducing chemotherapy efficiency. Different proteolytic enzymes, such as collagenase (Col) or bromelain, can directly attach to the surface of nanoparticles and improve their diffusion, but the method of ligation may also impair the enzymatic activity due to conformational changes or blockage of the active site. Herein, a "nanoenzyme capsule" was constructed by combining collagenase nanocapsules (Col-nc) with heavy-chain ferritin (HFn) nanocages encapsulating the chemotherapy drug doxorubicin (DOX) to enhance tumor penetration of the nanoparticles by hydrolyzing collagen from the ECM. Col-nc could protect the activity of the enzyme before reaching the site of action while being degraded under mildly acidic conditions in tumors, and the released proteolytic enzyme could digest collagen. In addition, HFn as a carrier could effectively load DOX and had a self-targeting ability, enabling the nanoparticles to internalize into cancer cells more effectively. From in vivo and in vitro studies, we found that collagen was effectively degraded by Col-nc/HFn(DOX) to increase the accumulation and penetration of nanoparticles in the solid tumor site and could alleviate hypoxia inside the tumor to enhance the antitumor effects of DOX. Therefore, the strategy of increasing nanoparticle penetration in this system is expected to provide a potential approach for the clinical treatment of solid tumors.

Cryopreserved clumps of mesenchymal stem cell/extracellular matrix complexes retain osteogenic capacity and induce bone regeneration.

BACKGROUND: Three-dimensional (3D) cultured clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. C-MSCs can regulate cellular functions in vitro and can be grafted into a defect site without an artificial scaffold to induce bone regeneration. Long-term cryopreservation of C-MSCs, which can enable them to serve as a ready-to-use cell preparation, may be helpful in developing beneficial cell therapy for bone regeneration. Therefore, the aim of this study was to investigate the effect of cryopreservation on C-MSCs. METHODS: MSCs isolated from rat femurs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make a round clumps of cells. The C-MSCs were cryopreserved in cryomedium including 10% dimethyl sulfoxide. RESULTS: Cryopreserved C-MSCs retained their 3D structure and did not exhibit a decrease in cell viability. In addition, stem cell marker expression levels and the osteogenic differentiation properties of C-MSCs were not reduced by cryopreservation. However, C-MSCs pretreated with collagenase before cryopreservation showed a lower level of type I collagen and could not retain their 3D structure, and their rates of cell death increased during cryopreservation. Both C-MSC and cryopreserved C-MSC transplantation into rat calvarial defects induced successful bone regeneration. CONCLUSION: These data indicate that cryopreservation does not reduce the biological properties of C-MSCs because of its abundant type I collagen. More specifically, cryopreserved C-MSCs could be applicable for novel bone regenerative therapies.

Proanthocyanidins-Loaded Nanoparticles Enhance Dentin Degradation Resistance.

Previous studies reported that grapeseed extract (GSE), which is rich in proanthocyanidins (PAs), improves the biodegradation resistance of demineralized dentin. This study aimed to investigate the effect of a new GSE delivery strategy to demineralized dentin through loading into biodegradable polymer poly-[lactic-co-glycolic acid] (PLGA) nanoparticles on the biodegradation resistance in terms of structural stability and surface/bulk mechanical and biochemical properties with storage time in collagenase-containing solutions. GSE-loaded nanoparticles were synthetized by nanoprecipitation at PLGA/GSE (w/w) ratios of 100:75, 100:50, and 100:25 and characterized for their morphological/structural features, physicochemical characteristics, and drug loading, entrapment, and release. Nanoparticle suspensions in distilled water (12.5% w/v) were applied (1 min) to demineralized dentin specimens by simulating pulpal pressure. The nanoparticle delivery was investigated by scanning electron microscopy (SEM)/transmission electron microscopy (TEM), and the GSE release from the delivered nanoparticles was further characterized. The variations in surface and bulk mechanical properties were characterized in terms of reduced elastic-modulus, hardness, nanoindentation testing, and apparent elastic-modulus with a storage time up to 3 mo. Hydroxyproline release with exposure to collagenase up to 7 d was estimated. An etch-and-rinse dentin adhesive was applied to investigate the morphology of the resin-dentin interface after nanoparticle delivery. Treatment with the GSE-loaded nanoparticles enhanced the collagen fibril structural resistance, reflected from the TEM investigation, and improved the biomechanical and biochemical stability of demineralized dentin. Nanoparticles having PLGA/GSE of 100:75 (w/w) showed the highest cumulative GSE release and were associated with the best improvement in biodegradation resistance. TEM/SEM showed the ability of the nanoparticles to infiltrate dentinal tubules' main and lateral branches. SEM revealed the formation of a uniform hybrid layer and well-formed resin tags with the presence of numerous nanoparticles located within the dentinal tubules and/or attached to the resin tag. This study demonstrated the potential significance of delivering collagen crosslinkers loaded into biodegradable polymer nanoparticles through the dentinal tubules of demineralized dentin on the biodegradation resistance.

bFGF and Activin A function to promote survival and proliferation of single iPS cells in conditioned half-exchange mTeSR1 medium.

Human induced pluripotent stem (iPS) cells can be well maintained by clonal growth. The pluripotent growth of single iPS cells is limited by low survival. To facilitate robust single iPS cells cultured in vitro, half-exchange mTeSR1 medium (HM), whole-exchange medium (WM) and iPS cell-derived conditioned medium (iPS-CM) culture were used. The effects of bFGF and Activin A on the growth of single iPS cells were explored. The dissociation and propagation of single iPS cells also included Accutase enzymatic isolation, Rho-associated protein kinase (ROCK) inhibitor Y27632 protection and high-density single-cell seeding (1 × 10(6) cells/well). CCK-8 assays demonstrated that the viability of clonal iPS cells in mTeSR1 medium and single iPS cells in HM, iPS-CM or WM supplemented with 100 ng/ml bFGF and 10 ng/ml Activin A was significantly higher than that in WM. Annexin v and propidium iodide (PI) assay, Calcein AM and EthD-III double staining also confirmed the similar results. ELISA assays showed that the levels of bFGF and Activin A of single iPS cells in HM and iPS-CM were higher than single iPS cells in WM. Meanwhile, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), quantitative Polymerase Chain Reaction (qPCR), Western Blotting (WB), Immunofluorescence (IF) and karyotype analysis revealed that HM culture was able to maintain undifferentiated markers of Nanog, Klf4, Sox2, Oct4, and did not affect the karyotype of iPS cells. Undifferentiated single iPS cells in HM displayed homogenized growth. These findings demonstrate that bFGF and Activin A are important for the survival and growth of single iPS cells. HM culture system combined Accutase, Y27632 and high-density single-cell seeding can facilitate short-term growth of single iPS cells in vitro.

Leukocyte invasion of the brain after experimental intracerebral hemorrhage in mice.

BACKGROUND AND PURPOSE: Neuroinflammatory processes contribute to secondary neuronal damage after intracerebral hemorrhage. We aimed to characterize the time course of brain immigration of different leukocyte subsets after striatal injection of either autologous blood or collagenase in mice. METHODS: Intracerebral hemorrhage was induced by injection of either autologous blood (20 µL) or collagenase (0.03 U) in C57Bl/6J mice. Hematoma volumetry was performed on cryosections. Blood volume was measured by hemoglobin spectrophotometry. Leukocytes were isolated from hemorrhagic hemisphere 1, 3, 5, and 14 days after intracerebral hemorrhage, stained for leukocyte markers, and measured by flow cytometry. Heterologous blood injection from CD45.1 mice was used to investigate the origin of brain-invading leukocytes. RESULTS: Collagenase injection induced a larger hematoma volume but a similar blood content compared with blood injection. Cerebral leukocyte infiltration in the hemorrhagic hemisphere was similar in both models. The majority of leukocytes isolated from the brain originated from the circulation. CD4+ T lymphocytes were the predominant brain leukocyte population in both models. However, cerebral granulocyte counts were higher after collagenase compared with blood injection. CONCLUSIONS: Brain infiltration of systemic immune cells is similar in both murine intracerebral hemorrhage models. The pathophysiological impact of invading leukocytes and, in particular, of T cells requires further investigation.

Dentin biomodification potential depends on polyphenol source.

Although proanthocyanidins (PACs) modify dentin, the effectiveness of different PAC sources and the correlation with their specific chemical composition are still unknown. This study describes the chemical profiling of natural PAC-rich extracts from 7 plants using ultra high pressure/performance liquid chromatography (UHPLC) to determine the overall composition of these extracts and, in parallel, comprehensively evaluate their effect on dentin properties. The total polyphenol content of the extracts was determined (as gallic acid equivalents) using Folin-Ciocalteau assays. Dentin biomodification was assessed by the modulus of elasticity, mass change, and resistance to enzymatic biodegradation. Extracts with a high polyphenol and PAC content from Vitis vinifera, Theobroma cacao, Camellia sinensis, and Pinus massoniana induced a significant increase in modulus of elasticity and mass. The UHPLC analysis showed the presence of multiple types of polyphenols, ranging from simple phenolic acids to oligomeric PACs and highly condensed tannins. Protective effect against enzymatic degradation was observed for all experimental groups; however, statistically significant differences were observed between plant extracts. The findings provide clear evidence that the dentin bioactivities of PACs are source dependent, resulting from a combination of concentration and specific chemical constitution of the complex PAC mixtures.

Improved enzymatic treatment for accurate cell counting from extracellular matrix-rich periodontal ligament cell sheets.

PURPOSE: The objective of this study was to establish a method for accurate cell counting from matrix-rich cell sheets in the clinical setting. MATERIALS AND METHODS: Human periodontal ligament (HPDL) cells were obtained from healthy donors to prepare PDL cell sheets. To obtain single cell suspensions, the cell sheets were treated with three different enzymatic formulations: collagenase alone, trypsin-ethylenediaminetetraacetic acid (EDTA) alone, and a combination of collagenase and trypsin-EDTA. After cell dispersion, cell numbers and cell survival rates were measured. To evaluate damage to the cell surfaces from the enzymes, the dispersed cells were analyzed by a flow cytometer with an anti-alkaline phosphatase antibody. RESULTS: Treatment with collagenase alone or trypsin-EDTA alone dispersed few cells from HPDL cell sheets. In contrast, combined treatment with collagenase and trypsin-EDTA successfully produced a large amount of single cells from cell sheets. Flow cytometry analysis showed that single cells obtained by combined use of collagenase and trypsin-EDTA preserved alkaline phosphatase epitopes on the cell surfaces. CONCLUSIONS: Cell sheets rich with extracellular matrix were dispersed via combined treatment with collagenase and trypsin-EDTA without destroying the expression of cell surface markers. The results suggest that this method would be useful for determining the accurate cell number of cell sheets for cell therapies and should also be applicable for other kinds of matrix-rich cell sheets.

Proanthocyanidins' efficacy in stabilizing dentin collagen against enzymatic degradation: MALDI-TOF and FTIR analyses.

OBJECTIVES: To investigate grape seed extract proanthocyanidins' (PA) capability in improving dentin collagen's sustainability in an enzymatic environment, given that the size and shape of the collagen samples, and the manner to apply PA are both clinically relevant. METHODS: Human dentin was sectioned into 6-µm-thick films. After demineralisation in 35wt% phosphoric acid for 15s, the films were subject to 30s of treatment at PA concentrations of 0% (control), 0.5%, 1%, 2%, 3.75%, 7.5% and 15% (w/w), respectively. The films were then digested in 0.1wt% collagenase for 1h and 24h. The amount of degraded collagen in the liquid digests was determined by MALDI-TOF mass spectroscopy. The trend of PA's incorporation into dentin collagen was analysed by ATR-FTIR. RESULTS: The control exhibited complete digestion in 1h. In contrast, collagen treated with 0.5% and 1% PA afforded 13.84±4.69% and an undetectable level of degradation, respectively in the first 1h of digestion, and additional 17.48±4.38% and 4.50±1.68%, respectively in the following 23h. Collagen treated with ≥2wt% PA was not significantly digested regardless of digestion time. FTIR spectroscopy revealed that PA incorporation was saturated at ≥2wt% PA. CONCLUSION: Thirty seconds of PA treatment at 2wt% and above could provide optimal protection for dentin collagen against collagenase digestion. CLINICAL SIGNIFICANCE: This study demonstrated PA's extraordinary efficiency in stabilizing demineralised dentin collagen when it is applied in a clinical relevant manner, and identified the optimal conditions for its utilization.

Effects of chemical cross-linkers on caries-affected dentin bonding.

The achievement of a strong and stable bond between composite resin and dentin remains a challenge in restorative dentistry. Over the past two decades, dental materials have been substantially improved, with better handling and bonding characteristics. However, little attention has been paid to the contribution of collagen structure/stability to bond strength. We hypothesized that the induction of cross-linking in dentin collagen improves dentin collagen stability and bond strength. This study investigated the effects of glutaraldehyde-and grape seed extract-induced cross-linking on the dentin bond strengths of sound and caries-affected dentin, and on the stability of dentin collagen. Our results demonstrated that the application of chemical cross-linking agents to etched dentin prior to bonding procedures significantly enhanced the dentin bond strengths of caries-affected and sound dentin. Glutaraldehyde and grape seed extract significantly increased dentin collagen stability in sound and caries-affected dentin, likely via distinct mechanisms.

Optimization of Ca(v)1.2 screening with an automated planar patch clamp platform.

INTRODUCTION: Ca(v)1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca(v)1.2 block is very important in developing drugs without cardiac liability. Ca(v)1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca(v)1.2 screening assay in PatchXpress 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca(v)1.2 antagonists and an agonist were tested. METHODS: HEK-293 cells stably transfected with hCa(v)1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba2+ and low magnesium intracellular recording solution to minimize rundown. Ca(v)1.2 currents were elicited from a holding potential of -60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout. RESULTS: Averaged peak Ca(v)1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 degrees C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg2+ from 2.7 mM to 0.2 mM and adding 100 nM Ca2+. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC(50) values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes. DISCUSSION: This assay can be used as a reliable pharmacological screening tool for Ca(v)1.2 block to assess compounds for cardiac liability during lead optimization.

Role of collagen fibers in acupuncture analgesia therapy on rats.

Acupuncture, a traditional Chinese therapeutic technique, has been put into practice for more than 4000 years and widely used for pain management since 1958. However, what is the mechanism underlying the acupuncture for analgesia effects by stimulation of acupoints, what substances receive the original mechanical acupuncture signals from the acupoints, or what transforms these signals into effective biological signals are not well understood. In this work, the role of collagen fibers at acupoints during acupuncture analgesia on rats was investigated. When the structure of the collagen fibers at Zusanli (ST36) was destroyed by injection of type I collagenase, the needle force caused by the acupuncture declined and the analgesic effects of rotation or lift-thrusting manipulations was attenuated accompanying the restraint of the degranulation ratios of mast cells. We propose that collagen fibers play an important role in acupuncture-induced analgesia, and they participate in signal transmission and transform processes.

Efficacy and mechanism of action of KHBJ-9B, a new herbal medicine, and its major compound triterpenoids in human cartilage culture and in a rabbit model of collagenase-induced osteoarthritis.

KHBJ-9B has been formulated by n-butanol fraction from 2 herbs known to have cartilage protection and anti-inflammatory effects. We elected to determine the osteoarthritic efficacy and mechanism of KHBJ-9B on human osteoarthritis cartilage explants culture and in a rabbit model of collagenase-induced osteoarthritis (CIA). The major chemical composition and quantification of KHBJ-9B was determined by high performance liquid chromatography. The efficacy of KHBJ-9B and its major compounds on cartilage protective effects such as inhibition of GAG release and type II collagen degradation, and their cytotoxicity in IL-1beta-treated human cartilage culture were examined. The mechanism of action of KHBJ-9B and its major compounds were evaluated by measuring inflammatory cytokines (IL-1beta and TNF-alpha) and matrix proteinases (ADAMTS-4, ADAMTS-5, MMP-1, MMP-13 and TIMP-3) in IL-1beta-treated human cartilage cultures. Also, the therapeutic effect of KHBJ-9B was confirmed using a collagenase-induced osteoarthritis (CIA) rabbit model. KHBJ-9B and 3 combined triterpenoids potently inhibited the release of proteoglycan and type II collagen in a dose dependent manner without cytotoxicity in IL-1beta-treated human cartilage explants culture, whereas its single major compounds (betulin, pimaradienoic acid and betulinic acid) and COX-2 inhibitor (NS398) showed little inhibition even at high concentrations. KHBJ-9B and the combination of 3 triterpenoids markedly inhibited the level of IL-1beta and TNF-alpha, and down-regulated the level of aggrecanases, ADAMTS-4, ADAMTS-5, MMP-1 and MMP-13, and up-regulated TIMP-3 in human cartilage explants culture. However, standard compounds and NS398 do not much affect the level of TNF-alpha, aggrecanases, and TIMP-3 in cartilage explants culture. In in vivo studies, KHBJ-9B significantly suppressed the stiffness level and global histologic score. Cartilage loss was significantly inhibited in the knee joint in a dose dependent manner, and this was associated with the finding that loss of proteoglycan, degradation of aggrecan and type II collagen was markedly reduced. These results suggest that the effect of KHBJ-9B is bigger than the effects of its single major compounds of triterpenoids or celecoxib inhibitors on cartilage protection and anti-inflammation in human cartilage and in in vivo model of osteoarthritis, and thus has potential for use in osteoarthritis treatment.

Comparison of the effects of collagenase and extract of Centella asiatica in an experimental model of wound healing: an immunohistochemical and histopathological study.

In this study, we compared the effects of collagenase and Centella asiatica in the rat model. Twenty-seven female rats were divided into three groups, and two full-thickness wounds were made for each animal. Collagenase ointment was applied topically to Group I and C. asiatica ointment to Group II rats. In Group III, no treatment was applied. On the third day of treatment, wounds on the left side of three animals of each group were excised. On the fifth and eighth day of the treatments, the same procedure was performed for the remaining animals. Indirect immunohistochemical examination was performed to detect transforming growth factor beta (TGF)-beta, endothelial and inducible nitric oxide synthase (eNOS and iNOS), vascular endothelial growth factor, TGF-alpha, laminin, fibronectin, collagen I, and interleukin-1beta. According to the measurements of the wound areas and wound healing periodo, collagenase was superior to the control group. Immunohistochemical examinations showed strong (+++) iNOS and TGF-beta immunoreactivities in C. asiatica group. eNOS immunoreactivity was moderate (++) in this group. For the collagenase group, iNOS, eNOS, and TGF-beta immunoreactivities were moderate (++). In the collagenase group, while TGF-beta and iNOS immunoreactivities were weaker, laminin and fibronectin reactivities were stronger than in C. asiatica and control groups. Collagenase was superior to C. asiatica according to the immunohistochemical findings. Collagenase ointment significantly improves the quality of wound healing and scar formation and is a more appropriate treatment choice than extract of C. asiatica in the early stages of the wound healing process.

Neurotization improves contractile forces of tissue-engineered skeletal muscle.

Engineered functional skeletal muscle would be beneficial in reconstructive surgery. Our previous work successfully generated 3-dimensional vascularized skeletal muscle in vivo. Because neural signals direct muscle maturation, we hypothesized that neurotization of these constructs would increase their contractile force. Additionally, should neuromuscular junctions (NMJs) develop, indirect stimulation (via the nerve) would be possible, allowing for directed control. Rat myoblasts were cultured, suspended in fibrin gel, and implanted within silicone chambers around the femoral vessels and transected femoral nerve of syngeneic rats for 4 weeks. Neurotized constructs generated contractile forces 5 times as high as the non-neurotized controls. Indirect stimulation via the nerve elicited contractions of neurotized constructs. Curare administration ceased contraction in these constructs, providing physiologic evidence of NMJ formation. Histology demonstrated intact muscle fibers, and immunostaining positively identified NMJs. These results indicate that neurotization of engineered skeletal muscle significantly increases force generation and causes NMJs to develop, allowing indirect muscle stimulation.

A novel method of isolation, preservation, and expansion of human corneal endothelial cells.

PURPOSE: To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs). METHODS: Human corneal Descemet's membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to 16 hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve growth factor (NGF), or basic fibroblast growth factor (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67. RESULTS: Digestion with collagenase A, but not Dispase, of the stripped Descemet's membrane generated HCEC aggregates, which preserved cell-cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell-cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell-cell junctions. CONCLUSIONS: Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.

Immunostimulation effect of jellyfish collagen.

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 degrees C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles' tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.