Pharmacological safety of dimethoxy curcumin-human serum albumin conjugate for potential therapeutic purpose.

Medicinal properties of curcumin are widely published. Previously, researchers used curcuminoid mixture comprising three chemical forms, out of which, the highest quantity is the most active molecule-dimethoxy curcumin (DMC). Reduced bioavailability, poor aqueous solubility, and quick hydrolytic degradation of DMC have projected challenges limiting its therapeutic value. However, selective conjugation of DMC with human serum albumin (HSA) enhances drug stability and solubility by several folds. Studies using animal models demonstrated potential anti-cancer/anti-inflammatory effects of DMCHSA; both studies showed results of local administration in peritoneal cavity and rabbit knee joint. DMC has prospects as intravenous therapeutic agent because carrier is HSA. However, before in vivo testing, important preclinical data required are toxicological safety and bioavailability of soluble forms of DMC. This study evaluated absorption, distribution, metabolism, and excretion of DMCHSA. Imaging technology and molecular analysis proved bio-distribution. The study also assessed the pharmacological safety of DMCHSA in mice in terms of its acute and sub-acute toxicity, complying with regulatory toxicology. Overall, the study demonstrated the safety pharmacology of DMCHSA upon intravenous infusion. This is a novel study establishing the safety of highly soluble and stable formulation of DMCHSA, qualifying it for intravenous administration and further efficacy evaluation in suitable disease models.

Preclinical evaluation of hydrogel sealed fluropassivated indigenous vascular prosthesis.

Background & objectives: Polyethylene terephthalate (PET) graft, designed and developed at our institute for vascular reconstruction, is porous to promote optimal incorporation and neointima formation, requiring pre-clotting or biomodification by sealing the pores before implantation. The objective of this study was to characterize, test and perform preclinical evaluation of hydrogel (alginate dialdehyde cross-linked gelatin) sealed fluoropassivated PET vascular prosthesis in pig model, so as to avoid pre-clotting, for its safety and efficacy before employing the indigenous and less expensive graft for clinical use. Methods: Hydrogel sealed, fluoropassivated PET vascular prosthesis were tested for haemocompatibility and toxicity followed by small animal toxicology tests and in vivo experiments in pigs receiving implantation at thoracic aorta. All 33 animals received test as well as control grafts with a plan for phased explantation at 2, 12 and 26 weeks. All animals underwent completion angiogram at the end of procedure as well as before graft explantation. Results: Haemocompatibility tests for haemolysis and toxicity tests showed no adverse events in tested mice and rabbits. Completion angiogram showed intact anastamosis and patent graft in each animal in post-operative period and at explantation. Gross and histopathological examination showed well-encapsulated grafts, clean glistening neointima and no evidence of thrombus in both test and control grafts. Interpretation & conclusions: Hydrogel sealed, fluoropassivated PET vascular prosthesis was found non-toxic, haemocompatible and remained patent in in vivo studies at planned intervals.

Silk Fibroin-Based Bioengineered Scaffold for Enabling Hemostasis and Skin Regeneration of Critical-Size Full-Thickness Heat-Induced Burn Wounds.

Millions of people around the globe are affected by full-thickness skin injuries. A delay in the healing of such injuries can lead to the formation of chronic wounds, posing several clinical and economic challenges. Current strategies for wound care aim for skin regeneration and not merely skin repair or faster wound closure. The present study aimed to develop a bioactive wound-healing matrix comprising natural biomaterial silk fibroin (SF), clinical-grade human fibrin (FIB), and human hyaluronic acid (HA), resulting in SFFIBHA for regeneration of full-thickness burn wounds. A porous, hemostatic, self-adhesive, moisture-retentive, and biomimetic scaffold that promotes healing was the expected outcome. The study validated a terminal sterilization method, suggesting the stability and translational potential of the novel scaffold. Also, the study demonstrated the regenerative abilities of scaffolds using in vitro cell culture experiments and in vivo full-thickness burn wounds of critical size (4 cm × 4 cm) in a rabbit model. Under in vitro conditions, the scaffold enhanced primary dermal fibroblast adhesion and cell proliferation with regulated extracellular matrix (ECM) synthesis. In vivo, the scaffolds promoted healing with mature epithelium coverage involving intact basal cells, superficial keratinocytes, multilayers of keratohyalin, dermal regeneration with angiogenesis, and deposition of remodeled ECM in 28 days. The relative gene expression of the IL6 marker indicated transitions from inflammation to proliferation stage. In addition, we observed skin appendages and rete peg development in the SFFIBHA-treated wound tissues. Although wound closure was observed, neither negative (untreated/sham) nor positive (commercially available product; NeuSkin) control wounds developed skin appendages/rete pegs or native skin architecture. After 56 days, healing with organized ECM production enabled the recovery of mechanical properties of skin with higher tissue maturity in SFFIBHA-treated wounds. Thus, in a single application, the SFFIBHA scaffold proved to be an efficient biomimetic matrix that can guide burn wound regeneration. The developed matrix is a suture-less, hemostatic, off-the-shelf product for potential wound regenerative applications.

Pharmacokinetics and Pharmacodynamics of Avian Egg-Yolk Derived Pure Anti-Snake Venom in Healthy and Disease Animal-Model.

The absence of Fc receptor binding, cost-effective production potential in large quantities in pure form, and better storage stability of avian immunoglobulin (IgY) advocate its therapeutic use as anti-snake venom (ASV). This study develops pure anti-neurotoxin (ANT- IgY) by immunizing White Leghorn hens with Cobra and Krait venoms for demonstrating antigen-antibody binding in vitro/in vivo. The purified IgY from immunized egg-yolk showed immunoprecipitation in Ouchterlony's Double Diffusion (ODD) experiment. For characterizing ANT-IgY distribution and clearance pattern, the study utilized an enzyme-linked immunosorbent assay (ELISA) in serum at different intervals following intravenous (IV) administration. The Kinetica 5.1 software estimated pharmacokinetic parameters, including half-life. The IgY showed a time-dependent elimination through the intestinal route in fecal matter. After conjugating with a fluorochrome-Vivotag-750S, injected the purified ANT-IgY intravenously into the healthy mice. Subsequently, captured live-animal images to demonstrate the distribution and elimination profile of the molecule. Intramuscular injection of fluorochrome-tagged venom created the envenomed mice model. The live-animal images demonstrated the quick mobilization of venom into vital tissues. Intravenous administration of tagged ANT-IgY in the envenomed model showed the movement of ASV to the tissues venom traffics. The observed pharmacological benefit promise scope of ASV-IgY for therapeutic use.

Advantages of hyaluronic acid as a component of fibrin sheet for care of acute wound.

Skin injury is followed by accumulation of a fibrin based provisional matrix which normally drives the process of wound repair. Exogenous fibrin with extra cellular matrix (ECM) components can also favor the wound healing process. In a preliminary study we found that lyophilized fibrin sheet (FS) arrest bleeding from rabbit skin wound but it remained dry during the repair period and did not accelerate the healing process better than untreated control. In the current study, hyaluronic acid (HA) was incorporated into FS and the resultant HA-FS promoted water retention and improved wound healing process. Gross-morphology, histopathology and histomorphometry were employed to compare qualitative and quantitative difference of wound healing in treated group against controls. In experimental sites (HA-FS), re-epithelialization was completed by 14 days with neo-vascularization and deposition of wavy bundles of collagen in the treated sites. Rate of healing process was different in treated and untreated wounds and most striking difference was the appearance of appendages, sebaceous gland and hair follicle at some locations in HA-FS treated sites. Therefore, HA with fibrin can create an effective wound care matrix which promotes water retention and wound healing potential.

Sustained release of curcumin from fibrin matrix induces cancer cell death and immunomodulation.

Despite the role of curcumin in controlling inflammation, angiogenesis, and cancer in human cells, its therapeutic use is limited. The reasons are quick metabolic breakdown, low aqueous solubility, and bioavailability. This study describes the advantages of clinical-grade curcumin-incorporated fibrin matrix either in lyophilized off-the-shelf wafer or injectable hydrogel forms, as a biodegradable local delivery system. To produce the curcumin-fibrin wafer, used clinical-grade fibrin sealant in a modified composition. To fabricate wafer, we premixed the curcumin with either fibrinogen or thrombin, before clotting into a hydrogel. Sustained release of active curcumin from fibrin wafer, suspended in culture medium at 37 °C lasted for seven days. Upon premixing albumin with thrombin and subsequently adding curcumin into the mixture improved the loading concentration and stability. Dose- and time-dependent apoptotic function of curcumin on cancer cell lines upon release from fibrin wafer, were demonstrated in vitro. In vivo immuno-modulation and a nontoxic response to curcumin released from fibrin into the peritoneal cavity of mice were established. The cytotoxic effect of released curcumin was demonstrated; showing both a preventive and therapeutic role against tumor growth. In vivo studies used Dalton's Lymphoma Ascites (DLA) mice model. Both implanted fibrin wafer and injected hydrogel can breakdown by a physiological process and get cleared by the fibrinolytic mechanism. The lyophilized fibrin wafer could function as a hemostat, adhere to surgical cancer tissues, and arrest bleeding. The potential of curcumin in preventing solid tumor metastasis may be explored upon the sustained delivery of the molecule from the fibrin wafer.

Refinement of the spinal cord injury rat model and validation of its applicability as a model for memory loss and chronic pain.

BACKGROUND: Laminectomy produces trauma in spinal cord injury (SCI) animal models resulting in impinging artefacts and welfare issues. Mechanizing laminectomy using a dental burr assisted (DBA) technique to reduce the impact of conventionally performed laminectomy on animal welfare without any alterations in the outcome of the model was previously demonstrated. However, further validation was necessary to establish it as an alternative in developing SCI rats as a model of chronic pain and memory loss. NOVEL METHOD: DBA technique was employed to perform laminectomy at T10-T11 vertebrae in rats undergoing contusion SCI as a model of chronic pain and memory loss. In a 56-day study, 24 female Wistar rats (Crl: WI) were assigned randomly to four equal groups: conventionally laminectomised, DBA laminectomised, conventionally laminectomised with SCI and DBA laminectomised with SCI. RESULTS: The study revealed DBA technique to cause less surgical bleeding (p = 0.001), lower Rat Grimace Scale (p = 0.0006); resulted in better body weight changes (p = 0.0002 on Day 7 and p = 0.0108 on Day 28) and dark phase activity (p = .0.0014 on Day 1; p = 0.0422 on Day 56). Different techniques did not differ in Basso Beattie Bresnahan score, novel object recognition, mechanical allodynia, number of surviving neurons and the area of vacuolation- indicating that the new method doesn't affect the validity of the model. COMPARISON WITH EXISTING METHODS: In comparison with the conventional technique, motorised laminectomy can be a valid tool that evokes lesser pain and ensures higher well-being in rats modelled for chronic pain and memory loss. CONCLUSIONS: The intended outcome from the model is not influenced by techniques whereas the DBA-technique is a refined alternative to the conventional method in achieving better welfare in SCI studies.

Optimizing fibrin hydrogel toward effective neural progenitor cell delivery in spinal cord injury.

Transplantation of neural progenitor cell (NPC) possessing the potential to differentiate into neurons may guard against spinal cord injury (SCI)- associated neuronal trauma. We propose that autologous-like NPC may reduce post-transplant immune response. The study used the rat SCI model to prove this concept. For isolation and expansion of rat NPC for cell-based SCI therapy, thein vitroprotocol standardized with human NPC seemed suitable. The primary aim of this study is to select a cell/neural tissue-compatible biomaterial for improving NPC survivalin vivo. The composition of the fibrin hydrogel is adjusted to obtain degradable, porous, and robust fibrin strands for supporting neural cell attachment, migration, and tissue regeneration. This study employed NPC culture to evaluate the cytocompatibility and suitability of the hydrogel, composed by adding graded concentrations of thrombin to a fixed fibrinogen concentration. The microstructure evaluation by scanning electron microscope guided the selection of a suitable composition for delivering the embedded cells. On adding more thrombin, fibrinogen clotted quickly but reduced porosity, pore size, and fiber strand thickness. The high activity of thrombin also affected NPC morphology and thein vitrocell survival. The selected hydrogel carried viable NPC and retained them at the injury site post-transplantation. The fibrin hydrogel played a protective role throughout the transfer process by providing cell attachment sites and survival signals. The fibrin and NPC together regulated the immune response at the SCI site reducing ED1+ve/ED2+vemacrophages in the early period of 8-16 d after injury. Migration ofß-III tubulin+veneural-like cells into the fibrin-injected control SCI is evident. The continuous use of a non-neurotoxic fibrin matrix could be a convenient strategy forin vitroNPC preparation, minimally invasive cell delivery, and better transplantation outcome.

Exploitation of fibrin-based signaling niche for deriving progenitors from human adipose-derived mesenchymal stem cells towards potential neural engineering applications.

Adipose-derived mesenchymal stem cells (hADMSC) retaining proliferation and multi-differentiation potential may support the central nervous system (CNS) regeneration. Multipotency of MSC may result in both desirable and undesirable cells, post-transplantation. A better strategy to attain desired cells may be in vitro commitment of hADMSCs to uni-/bi- potent neural progenitor cells (NPCs), prior to transplantation. Derivation of stable NPCs may require a suitable niche eliciting proliferation and differentiation signals. The present study designed a biomimetic niche comprising insoluble fibrin supported adhesion matrix and exogenously added growth factors (GFs) for deriving different neural cells and established the role of Notch and Wnt signals for proliferation and differentiation of hADMSCs, respectively. The stable transformation of hADMSCs into neurospheres (NS) comprising Nestin+ve NPCs was achieved consistently. Slight modifications of niche enable differentiation of NS to NPCs; NPCs to neurons; NPCs to oligodendrocyte progenitor cells (OPCs); and OPCs to oligodendrocytes (OLG). Fibrin plays a crucial role in the conversion of hADMSC to NS and NPCs to OPCs; but, not essential for OPC to OLG maturation. Co-survival and cell-cell interaction of NPC derived neurons and OPCs promoting OLG maturation is illustrated. The designed biomimetic niche shows the potential for directing autologous ADMSCs to neural cells for applications in regenerative medicine.

Generation of niche tuned antifibrotic fibroblasts and non-viral mediated endothelial commitment using adipose stem cells for dermal graft development.

Cell-based skin substitute generation has seen considerable development. Combining synthetic scaffolds with biomimetic fibrin does direct both exogenous and endogenous stem cell differentiation, addressing needs for reliable tissue engineering. However, lack of immediate vasculature within implantable grafts remains critical for its sustenance and integration. Multipotency, high proliferation potential, ability to release multiple growth factors (GFs), and autologous availability highlight the use of human adipose derived mesenchymal stem cells (hADMSCs) in tissue-engineered dermal grafts (TEDG) construction. However, hADMSCs' insufficiency to independently establish angiogenesis within tissue constructs demands improvement of stem cell application for dermal graft survival. Approaches to harness microenvironmentally sensitive paracrine interactions could improve the angiogenic efficiency of hADMSCs within TEDG. This study conceptualized a fibrin-based niche, to direct hADMSCs toward a nonfibrotic fibroblast commitment and incorporation of bioengineered hADMSCs, specifically releasing potent angiogenic factors within TEDG. Coexistence of tuned fibroblast and endothelial lineage committed cells contributed to well-regulated extracellular matrix formation and prevascularization. Adequate cell proliferation; sustained transient release of angiogenic GFs till 20 days; directed dermal, endothelial, fibroblast, and vascular smooth muscle cell differentiation; and favored elastin and collagen deposition were achieved in vitro. In conclusion, specific niche composition and employment of bioengineered hADMSCs favor implantable TEDG construction.

Human-Derived Scaffold Components and Stem Cells Creating Immunocompatible Dermal Tissue Ensuing Regulated Nonfibrotic Cellular Phenotypes.

Regeneration of large-sized acute and chronic wounds provoked by severe burns and diabetes is a major concern worldwide. The availability of immunocompatible matrix with a wide range of regenerative medical applications, more specifically, for nonhealing chronic wounds is an unmet clinical need. Extrapolating the in vitro tissue engineering knowledge for in vivo guided wound regeneration could be a meaningful approach. This study aimed to develop a completely human-derived and minimally immune-responsive scaffold comprising of acellular amniotic membrane (AM), fibrin (FIB) and hyaluronic acid (HA), termed AMFIBHA. The potential for in vivo guidance of skin regeneration was validated through in vitro dermal tissue assembly on the combination scaffold by growing human fibroblasts, differentiated from human adipose tissue-derived mesenchymal stem cells (hADMSCs). An effective method was standardized for obtaining decellularized amnion (dAM) for assuring better immuno-compatibility. The biochemical stability of dAM upon plasma sterilization (pdAM) confirms its suitability for both in vitro and in vivo tissue engineering. The problem of poor handling characteristics was solved by combining the dried dAM with fibrin derived from a clinically used fibrin sealant kit. An additional constituent HA, derived from human umbilical cord tissue, imparts the required water absorption and retention property for better cell migration and growth. Post sterilization, the combination scaffold AMFIBHA demonstrated hemo-/cytocompatibility, confirming the absence of detergent residuals. Upon long-term (20 days/40 days) culture of hADMSC-derived fibroblasts, the suppleness of generated tissue was established by demonstrating regulated deposition of collagen, elastin, and glycosaminoglycans using both qualitative and quantitative measurements. Regulated expressions of transforming growth factors-beta 1 (TGF-ß1) & TGF-ß3, alpha smooth muscle actin (α-SMA), fibrillin-1, collagen subtypes, and elastin suggest non-fibrotic fibroblast phenotype, which could be an effect of microenvironment endowed by the AM, FIB, and HA. In burn wound model experiments, immune response to cellular AM was prominent as compared to untreated/sham control wounds and decellularized AM-treated and AMFIBHA-treated wounds, ensuring biocompatibility. Wound regeneration with complete epithelialization, angiogenesis, development of rete pegs, and other skin appendages were clearly visualized in 28 days after treating large-sized (4 × 4 cm2), debrided, full-thickness third-degree burn wounds, indicating guided wound regeneration potential of AMFIBHA dermal substitute.

Design of fibrin matrix composition to enhance endothelial cell growth and extracellular matrix deposition for in vitro tissue engineering.

Tissue-engineered blood vessel substitutes should closely resemble native vessels in terms of structure, composition, mechanical properties, and function. Successful cardiovascular tissue engineering requires optimization of in vitro culture environment that would produce functional constructs. The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system to render elasticity to blood vessel wall, whereas collagen is responsible for providing mechanical strength. The objective of this study was to understand the significance of various ECM components on endothelial cell (EC) growth and tissue generation. We demonstrate that, even though fibrin is a good matrix for EC growth, fibronectin is the crucial component of the fibrin matrix that enhances EC adhesion, spreading, and proliferation. Vascular EC growth factor is known to influence in vitro growth of EC, but, so far, ECM deposition in in vitro culture has not been reported. In this study, it is shown that incorporation of a mixture of hypothalamus-derived angiogenic growth factors with fibrin matrix enhances synthesis and deposition of insoluble elastin and collagen in the matrix, within 10 days of in vitro culture. The results suggest that a carefully engineered fibrin composite matrix may support EC growth, survival, and remodeling of ECM in vitro and impart optimum properties to the construct for resisting the shear stress at the time of implantation.

Differentiation of circulating neural progenitor cells in vitro on fibrin-based composite -matrix involves Wnt- ß-catenin-like signaling.

Isolation of progenitors with regenerative potential and their in vitro induction to specific lineage may be necessary for effective cell transplantation outcome. Earlier, we standardized specific niche for derivation of neural progenitor cells (NPCs) from circulating mononuclear cells to neural like cells (NLC) in vitro, for applications in neural regeneration. The current study analysed the prospective involvement of signaling mechanism for in vitro lineage commitment of circulating NPCs. Preferred mechanism selected was Wnt-like signaling because this is one of the pathways implicated in the central nervous system (CNS) development and homeostasis. We sought to determine the activation of Wnt3a-specific genes in the standardized NPC culture system. To start with, it was found that when standardized NPC culture niche was supplemented with Wnt 3a protein, no additional morphological changes happen. Chemical inhibitors of the pathway retarded NPC to NLC conversion both in the absence and presence of supplemented Wnt-3a. In earlier studies, involvement of the niche constituents- fibronectin (FN), laminin (La) and fibrin (Fib)- for NPC growth and differentiation was established. In an attempt to study the role of these adhesive proteins by adding antibodies against FN, La & Fib together, molecular level signaling changes seen were comparable to that occur in response to Wnt3a chemical inhibitor. Therefore, induction of Wnt 3a-like signal from the matrix-dependent niche constituents may be implicated in the differentiation of NPC to NLC. The results substantiate the potential applications of the fibrin-based composite niche in neural engineering for regeneration.

Engineered Human Adipose-Derived Stem Cells Inducing Endothelial Lineage and Angiogenic Response.

IMPACT STATEMENT: With respect to the persistent hunt for a cytocompatible, translational, reproducible, and effective approach in engineering primary human adipose-derived mesenchymal stromal cells (hADMSCs), we demonstrate the application of Neon® Transfection System in adequate transient delivery of angiogenic factors. The study presents functional assessment of this approach in vitro, with two notable outcomes at translational perspective; (1) Bioengineered hADMSCs secretome does induce endothelial lineage commitment of stem cells at both transcriptional and translational levels and (2) Combinatorial delivery of vascular endothelial growth factor A and hypoxia-inducible factor-1α by bioengineered hADMSCs enhance upregulation of endothelial cell proliferation, migration-associated wound closure, and endothelial tube formation with augmented Flk-1 expression, as compared with their independent actions. The methods described in this study paves way for in vivo evaluation on identification of appropriate chronic wound models and subsequently for clinical translation. The technology developed also has application in vascularization of tissue-engineered constructs.

A novel technique to develop thoracic spinal laminectomy and a methodology to assess the functionality and welfare of the contusion spinal cord injury (SCI) rat model.

This study reports the advantage of a novel technique employing a motorised dental burr to assist laminectomy over the conventional manual technique at T10-T11 vertebra level in a rat model of spinal cord injury. Twenty-four female rats were randomly assigned to four groups: (1) conventionally laminectomised, (2) dental burr assisted laminectomised, (3) conventionally laminectomised with spinal cord contusion and (4) dental burr assisted laminectomised with spinal cord contusion. Basso Beattie Bresnahan (BBB) score, postoperative body weights, rat grimace scale (RGS), open cage activity and rearing was studied at 1, 7, 14, 21 and 28 days postoperatively, and area of spinal tissue affected was evaluated histologically. Laminectomised and spinal cord injured rats from dental burr groups showed significantly more weight gain and less weight loss respectively in comparison with respective conventionally laminectomised groups at various time points. Significantly higher RGS score was noticed in conventionally laminectomised animals on Day 1 in comparison to burr assisted laminectomy and presence of pain was evident until Day 7 in the conventionally spinal cord injured group. BBB score did not differ between techniques, whereas laminectomy groups showed more resting time than spinal injury groups. High rearing score was significantly higher in groups which underwent dental burr assisted technique at various time points with respect to their conventional counterparts. This study suggests that the use of dental burr assisted technique to perform laminectomy will bring refinement by producing less pain, aiding in better recovery, removing procedural artefacts without affecting the outcome of the model.

Regulation of endothelial cell phenotype by biomimetic matrix coated on biomaterials for cardiovascular tissue engineering.

One major weakness that all cardiovascular replacements have in common is the lack of endothelial cell (EC) growth and post-implant remodeling of the device. The emerging field of tissue engineering focuses on the in vitro generation of functional organ replacements using living endothelial cells and other vascular cells for which nondegradable or biodegradable scaffold base materials are used. In this paper, it is demonstrated that some of the cardiovascular device materials in clinical use lack the ability to promote endothelial cell growth in vitro. We previously established a biomimetic matrix composition which supports the growth of human umbilical vein endothelial cells (HUVECs) while maintaining normal physiology in vitro. Here the effectiveness of the same coating to preserve the normal antithrombotic phenotype of endothelial cells grown on biomaterials was evaluated. The up/down-regulation of two prothrombotic and two antithrombotic molecules by HUVECs grown on bare material surfaces were compared with that on composite-coated materials. The suitability of this approach for blood-contacting applications was investigated by in vitro blood compatibility studies as recommended in ISO10993 part 4, by putting an EC-seeded surface in contact with human whole blood. It is demonstrated that EC-seeded bare material surfaces are prothrombotic, whereas surfaces pre-coated with biomimetic molecules facilitated maintenance of the normal EC phenotype and reduced the risk of platelet adhesion and activation of blood coagulation. The results presented here suggest that matrix composed of biomimetic adhesive proteins and growth factors is suitable for cardiovascular tissue engineering to improve biological function, irrespective of the material chosen to meet the mechanical properties of the device.

Growth factors upregulate deposition and remodeling of ECM by endothelial cells cultured for tissue-engineering applications.

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial for their long-term function. The interaction between cells and extra-cellular components is indispensable in determining cellular behavior in tissues and on biomaterials. The fibrin that contains fibronectin shows promise in most aspects as a tissue engineering scaffold, whereas, deposition of elastin and collagen by endothelial cells grown in the lumen of the construct is desirable to improve post implant retention, mechanical stability and vaso-responsiveness. So far there is no report on production of extra-cellular matrix (ECM) proteins, elastin and collagen by endothelial cells (EC) in in vitro culture conditions. In this study, we have used a biomimetic approach of providing multiple growth factors (GF) in the fibronectin (FN)-containing fibrin matrix to induce production of elastin and collagen by the endothelial cells for application in vascular tissue engineering. Deposition of elastin and collagens with matrix remodeling is demonstrated through qualitative analysis of the matrices that were recovered after growing cells on the initial fibrin-FN-GF matrix. Expressions of mRNA for both proteins were assessed by real time polymerase chain reaction (RT-PCR) to estimate the effects of multiple growth factor compositions. Marked deposition of elastin and collagen was evidenced by staining the recovered matrix after different culture intervals. Obviously, the biomimetic environment created by adding angiogenic and platelet growth factors in the fibrin-fibronectin-gelatin matrix can induce deposition of collagens and elastin by EC.

Increased platelet cholesterol and decreased percentage volume of platelets as a secondary risk factor for coronary artery disease.

Platelet hyperactivity is likely to contribute to the progression of atherogenesis and organized thrombus formation on vascular surfaces. The purpose of this study was to examine the effect of hypercholesterolemia on the cholesterol content of platelets, on platelet responsiveness and other platelet indices using platelets from 5 groups of age-matched subjects (n = 30 each), which includes healthy controls. All groups except controls had a high plasma lipid profile. While subjects in group I had only hyperlipidemia, those in groups II and III had hyperlipidemia in conjunction with diabetes mellitus and hypertension, respectively. The fourth group consisted of patients with confirmed coronary artery disease (CAD). The parameters studied include packed cell volume of platelets (platelet crit), platelet distribution width (PDW), platelet cholesterol and platelet aggregation in response to adenosine diphosphate and collagen. All the patient groups showed increased platelet aggregation (p < 0.05) and low platelet crit compared with controls (p < 0.05). In addition, platelet cholesterol was increased in patients with coronary disease, hyperlipidemia and diabetes mellitus (p < 0.05) but not in patients with hypertension (p > 0.05); PDW was high only in CAD (p < 0.05). A higher PDW indicated a prothrombotic tendency in CAD patients. Our data suggest that hyperlipidemia increases the lipid content in platelets and enhances their reactivity. Hyperactive platelets with increased platelet cholesterol may contribute to accelerated atherogenesis associated with CAD.

A biochemical study on the effect of proteolysis of beta-thromboglobulin proteins released from activated platelets on fibroblast proliferation.

beta-Thromboglobulin (beta-TG) proteins are a heterogeneous group released from platelet alpha-granules on activation and have an effect on fibroblast migration and proliferation. We have previously reported the action of a metal-dependent protease on platelet-released proteins, which generates low-molecular-weight proteins that could be inhibited by ethylenediaminetetraacetic acid (EDTA). To understand the physiological significance of the breakdown of proteins after release, their effect on fibroblast proliferation in vitro was studied. Platelet releasates were obtained without and with EDTA inhibition designated as R1 and R2, respectively, and proteins were affinity purified for testing. Cell proliferation was measured using [(3)H]-thymidine assay. Both R1 and R2 showed maximum activity at 100 microg/ml and R2 elicited significant proliferation compared to R1. When affinity-purified proteins were tested at 100 ng/ml, high-molecular-weight proteins showed significantly higher proliferation than low-molecular-weight proteins. We have shown that beta-TG is cleaved after being released from activated platelets, thereby becoming less mitogenic for fibroblasts.

Bio-mimetic composite matrix that promotes endothelial cell growth for modification of biomaterial surface.

The incidence of thrombogenesis and occlusion of cardiovascular implants is likely to be reduced by endothelial cell (EC) growth promotion on biomaterials used for device fabrication. However, proper signaling between the matrix proteins deposited on the device surface and the cells grown on it is a prime requirement for growth and function. It was demonstrated earlier that a composition of matrix proteins that include fibrin, fibronectin, gelatin, and growth factors maintain a steady proliferation potential and prolong the survival of endothelial cells in vitro. In this study, assessment of the same matrix to prevent EC from dedifferentiation during in vitro culture and to promote endothelialization of biomaterials used for fabrication of cardiovascular implants is carried out. Up/down regulation of m-RNA expression for a prothrombotic molecule-plasminogen activator inhibitor (PAI), and two antithrombotic molecules- nitric oxide synthetase (eNOS) and tissue plasminogen activator (t-PA) are chosen as the indicators of cell dedifferentiation during cell culture and passaging. Immunostaining for vinculin and actin demonstrated that composite coating on biomaterials improves focal adhesion and cytoskeletal organization that increases the quality of EC grown on it. EC proliferation, measured by (3)H-thymidine uptake, on all bare materials was poor and high incidence of cell apoptosis was noticed within 72 h in culture, whereas once coated with composite all materials showed good proliferation and survival. The results suggest that the designed composition of biomimetic adhesive proteins and growth factors is suitable for EC growth, survival, and functional integrity, thus making it suitable for cardiovascular tissue engineering that requires in vitro EC culture.