Search

The many studies conducted so far reveal that Tissue Engineering of the skin is only at the beginning of its use in human applications. Burns patients were the first targets for such tissue substitutes, then chronic diseases, such as venous ulcers, have followed. The more experience is gained from the surgeon, the more feedback for the basic scientist to improve the product and to broaden clinical indications. Nowadays, progress in cell culture and biomedical material technologies have added two important spare parts: epidermis and dermis to the surgeon's toolbox which can be reconstituted in the laboratory from small biopsies of the same recipient. Other parts will follow in a few years, with the final aim to generate a full transplantable replica of the skin with adnexa and vasculature.

The objective of this preliminary study was to develop a reabsorbable vascular patch that did not require in vitro cell or biochemical preconditioning for vascular wall repair. Patches were composed only of hyaluronic acid (HA). Twenty male Wistar rats weighing 250–350 g were used. The abdominal aorta was exposed and isolated. A rectangular breach (1 mm × 5 mm) was made on vessel wall and arterial defect was repaired with HA made patch. Performance was assessed at 1, 2, 4, 8, and 16 weeks after surgery by histology and immunohistochemistry. Extracellular matrix components were evaluated by molecular biological methods. After 16 weeks, the biomaterial was almost completely degraded and replaced by a neoartery wall composed of endothelial cells, smooth muscle cells, collagen, and elastin fibers organized in layers. In conclusion, HA patches provide a provisional three-dimensional support to interact with cells for the control of their function, guiding the spatially and temporally multicellular processes of artery regeneration. © 2011 Wiley-Liss, Inc. Microsurgery, 2011.

A significant amount of recent interest has been focused on the possibility that adult human stem cells are a realistic therapeutic alternative to embryonic stem cells. Multipotent stem cells that have characteristics reminiscent of embryonic neural crest stem cells have been isolated from several postnatal tissues, including skin, gut, dental pulp and the heart, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. In the present work, we investigated this issue with regard to skin-derived precursors (SKPs) and adipose-derived stem cells (ADSc)Adult stem cells isolated from skin and from adipose tissue derived from the same adult donor were treated with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Neurospheres obtained were first expanded and evaluated in term of proliferative ability, and then their neuronal differentiation potential was analysed.Adipose- and skin-derived neurospheres grew in suspension as spheres in the presence of the mitogens FGF2 and EGF. With this protocols, the spheres have been able to proliferate and to originate Schwann and glial-like cells.In summary, we have demonstrated in this work that multipotent adult precursor cell can be isolated and expanded from two accessible adult tissue sources: skin and adipose tissue. The work described in this paper provides the framework for our attempts to use SKPs or ADSc as autologous adult stem cell population for cell replacement and discovery research.

Atypical antipsychotics (APDs) are currently used in clinical practice for a variety of mental disorders such as schizophrenia, bipolar disorder and severe behavioral disturbances. A well-known disadvantage of using these compounds is a propensity for weight gain, resulting frequently in obesity. The mechanisms underlying pharmacologically induced weight gain are still controversial. The objective of this study was to evaluate in vitro the effects of different APDs on adipogenic events in cultured human pre-adipocytes and in rat muscle-derived stem cells (MDSCs), aiming to identify a common intracellular event contributable to these drugs. Culture behavior was evaluated in terms of cell proliferation, lipid accumulation, gene expression and morphological features. Results indicate that APDs influence adipogenic events through changes in the differentiation and proliferation of pre-adipocytes and MDSCs that are brought on by protein kinase C-β (PKC-β) activation. These data identify a signaling route that could be a potential target of pharmacological approaches for preventing the weight gain associated with APD treatment.

The present study describes the production of hyaluronan based porous microparticles by a semi-continuous gas anti-solvent (GAS) precipitation process to be used as a growth factor delivery system for in vivo treatment of ulcers. Operative process conditions, such as pressure, nozzle diameter and HYAFF11® solution concentrations, were adjusted to optimize particle production in terms of morphology and size. Scanning electron microscopy (SEM) and light scattering demonstrated that porous nano-structured particles with a size of 300 and 900 nm had a high specific surface suitable for absorption of growth factors from the aqueous environment within the polymeric matrix. Water acted as a plasticizer, enhancing growth factor absorption. Water contents within the HYAFF11® matrix were analyzed by differential scanning calorimetry (DSC). The absorption process was developed using fluorescence dyes and growth factors. Immunohistochemical analysis confirmed the high efficiency of absorption of growth factor and a mathematical model was generated to quantify and qualify the in vitro kinetics of growth factor release within the polymeric matrix. In vivo experiments were performed with the aim to optimize timed and focal release of PDGF to promote optimal tissue repair and regeneration of full-thickness wounds.

For peripheral nerve regeneration, three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance. The purpose of this study was to test in vitro a novel hyaluronic acid-based tubular conduit (HYAFF-11 biomaterials: 1 x 10 mm) as a nerve guide.Human fibroblasts, RN22 Schwann cell lines, human umbilical vein endothelial cells and primary nerve cells, obtained from neonatal rat sciatic nerve, were harvested and seeded on HYAFF-11 devices. Histologic (hematoxylin-eosin), immunohistochemical (antibodies to S100, CD31 and Von Willebrand factor) and PCR analyses were performed after 7 and 14 days from cell seeding onto biomaterials. MTT-based (thiazolyl blue) and DELFIA cell proliferation kit tests were performed to observe the biocompatibility of the cells cultured within the biomaterial devices.We concluded that the conduits were not cytotoxic and demonstrated that cultured RN22 Schwann cells and rat Schwann cells grow in vitro on new artificial nerve conduits. We thus inferred that the HYAFF-11 conduit was a suitable biomaterial able to support nerve cell growth in vitro and after 14 days of cultivation, remained circular with a round lumen, maintaining the size and shape of its original architecture. Finally, attachment and proliferation of endothelial cells attested to the feasibility of developing a coculture system to promote in vivo integration of a microvascularized nerve substitute.HYAFF-11 pre-seeded with Schwann and endothelial cells has the potential to be an alternative to autografting for the repair of long peripheral nerve defects.

Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCβ activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCβ siRNA silencing. The differentiated adipocytes, when implanted in vivo , form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus.

Few manuscripts describe the construction of an adipose tissue composite flap able to create an in vivo microenvironment and a neovasculature that can grow with and service implanted adipose tissue. Creation of an in vivo vascular carrier and tissue chamber for volume-stable transplanted adipose tissue was attempted using jejunum segments with intact circulation in 18 male Wistar rats. Intestinal segments were filled with autologous adipose tissue. Histologic (hematoxylin-eosin), immunohistochemical (antibodies to leptin and to vascular endothelial growth factor) and ultrastructural analyses were used to evaluate the results at 6, 18, and 60 days after surgery. Macroscopic observation confirmed the feasibility of this prefabricated adipose tissue flap: no loss of weight or volume occurred at any time point. Histologic analysis showed normal morphologic features of transplanted adipose tissue. Immunohistochemical studies confirmed the vitality of adipose tissue and the presence of a microvascular network within the flap. Small intestinal segments denuded of the mucosal layer can support in vivo transplanted adipose tissue.

In this study we coated a new biocompatible, nanostructured titanium alloy, Ti13Nb13Zr, with a thin layer of hydroxyapatite nanocrystals and we investigated the response of human bone-marrow-derived mesenchymal cells. The coating was realized using a slightly supersaturated CaP solution, which provokes a fast deposition of nanocrystalline hydroxyapatite. A thin layer of deposition is appreciable on the etched Ti13Nb13Zr substrates after just 1.5 h soaking in the CaP solution, and it reaches a thickness of 1-2 mum after 3 h soaking. The coating seems thinner than that deposited on Ti6Al4V, which was examined for comparison, likely because of the different roughness profiles of the two etched alloys, and it is constituted of elongated HA nanocrystals, with a mean length of about 100 nm. Mesenchymal stem cells were seeded onto coated and uncoated Ti alloys and cultured for up to 35 days. Cell morphology, proliferation and differentiation were evaluated. The cells display good adhesion and proliferation on the uncoated substrates, whereas the presence of hydroxyapatite coating slightly reduces cell proliferation and induces differentiation of MSCs towards a phenotypic osteoblastic lineage, in agreement with the increase of the expression of osteopontin, osteonectin and collagen type I, evaluated by means of rt-PCR. Type I collagen expression is higher in Ti13Nb13Zr MSC culture compared to Ti6Al4V, standing for a more efficient extracellular matrix deposition.

Tissue engineering is a promising approach to developing hepatic tissue suitable for the functional replacement of a failing liver. The aim of the present study was to investigate whether an extracellular cell matrix obtained from fibroblasts-cultured within scaffolds of hyaluronic acid (HYAFF™) could influence the proliferation rate and survival of rat hepatocytes both during long-term culture and after in vivo transplantation. Cultures were evaluated by histological and morphological analysis, a proliferation assay and metabolic activity (albumin secretion). Hepatocytes cultured in extracellular matrix-enriched scaffolds exhibited a round cellular morphology and re-established cell–cell contacts, growing into aggregates of several cells along and/or among fibers in the fabric. Hepatocytes were able to secrete albumin up to 14 days in culture. In vivo results demonstrated the biocompatibility of HYAFF-11 TM implanted in nude mice, in which hepatocytes maintained small well-organised aggregates until the 35th day. In conclusion, the presence of a fibroblast-secreted extracellular matrix improved the biological properties of the hyaluronan scaffold, favoring the survival and morphological integrity of hepatocytes in vitro and in vivo.

Experiences coming from many cell-culture studies has brought about the concept that tissue and organ reconstruction should be performed in a three-dimensional environment as it normally occurs in vivo. As far as endothelial cell culture is concerned, it has been shown that angiogenesis can be successfully achieved only when cells are cultured in the presence of collagen-based matrices or basal membrane substrates. The aim of the present investigation is to demonstrate that human umbilical vein endothelial cells (HUVEC) can be grown and differentiated on an artificial dermis obtained by fibroblasts cultured on hyaluronic acid-based scaffolds. For this purpose, we have cultured HUVEC, retrieved by collagenase digestion of perfused human umbilical vein either alone and with fibroblast at 1/1 ratio into HYAFF-11 non-woven mesh. Cultures were maintained for up to 3 weeks. Samples were taken at different time points within this period for the MTT proliferation test and for immunohistochemical analysis. Our results demonstrate that hyaluronan-based biomaterials (HYAFF-11 NW mesh) represent a suitable substrate for HUVEC adhesion, proliferation and reorganization in microcapillary network.

The objective of the present study was to demonstrate the safety and efficacy of a dermal replacement for cutaneous wounds of diverse origin. Autologous fibroblasts were cultured in fleece scaffolds made from benzyl esters of hyaluronic acid and applied onto cutaneous lesions. The cases presented are (1) skin removal for multiple epithelioma and (2) chronic deep decubitus ulcer. Dermal-like tissue applied by the surgeon elicited no adverse reactions, and was fully integrated and well-vascularized by 1-3 weeks. In Case 1, the material was fully integrated after 1 week, and after 3 weeks an epidermal autograft was overlaid which showed good take with excellent integration observed after 4 weeks. At 12 months, skin demonstrated visual normo-elastic properties and no signs of excessive scarring. In Case 2, 2-3 weeks after the dermal implant was applied, the wound was invaded with granulation tissue and healing occurred by secondary intention. The ulcer was healed by 8 weeks, with the biomaterial completely resorbed and a complete re-epithelialization over the dermal-like tissue. These results suggest that autologous fibroblast culture in hyaluronan-derived scaffolds may be successfully grafted in diverse cutaneous pathologies and constitute a suitable bed for further epidermal implantation.

Tissue engineering offers the possibility to reconstruct tissue substitutes in order to replace lost or damaged tissues. The availability of appropriate biomaterial devices is essential to allow in vitro cultured cells to behave as in the original tissues in vivo. In our studies we utilized a seminatural biomaterial made up by the benzyl ester of hyaluronan to grow keratinocytes, fibroblasts and chondrocytes. Keratinocytes and fibroblasts were isolated from human foreskin. Cells were separately cultured on two different hyaluronan based biomaterial devices for the first 15 days and then co-cultured for an additional period of 2 weeks. Keratinocytes gave rise to a well-differentiated epithelial layer, while fibroblasts were able to synthesize all the main extracellular molecules inside the biomaterial spaces, forming dermal-like tissues. When these two tissues were co-cultured, a skin equivalent was formed with a dermal–epidermal junction. Chondrocytes were obtained from chick-embryo sterna and cultured for 21 days inside a non-woven scaffolding made up of a hyaluronan-based biomaterial. Cells were able to organize themselves into nodules embedded in a dense metachromatic substance in which type II collagen was present. Data from this study suggest that this novel class of hyaluronan derived biomaterials is suitable for different cell culture and in vitro tissue reconstruction. © 1999 Kluwer Academic Publishers

Coverage of large, full-thickness burns presents a challenge for the surgeon due to the lack of availability of the patient's own skin. Currently, tissue engineering offers the possibility of performing a suitable therapeutic wound coverage after early burn excision by using cultured keratinocyte sheets supported by a dermal layer. The aim of this study was to develop and characterize a skin substitute composed of both epidermal and dermal elements. For this purpose we grew keratinocytes and fibroblasts separately for 15 days within two different types of biomaterials. Cells then were co-cultured for an additional period of 15 days, after which samples were taken and processed with either classic or immunohistochemical stainings. Results showed that (1) human fibroblasts and keratinocytes can be cultured on hyaluronic acid-derived biomaterials and that (2) the pattern of expression of particular dermal-epidermal molecules is similar to that found in normal skin. The data from this study suggest that our skin equivalent might be useful in the treatment of both burns and chronic wounds.

INTRODUCTION: A greater supply of tendinous tissue can be obtained through tissue engineering technology with increasing application of adult stem cells. It is well known that adipose-derived stem cells (ADSCs), found in abundance in adipose tissue, have the same differentiating capacity as mesenchymal stem cells yet have the advantage of being easily isolated. In the present study, we combined the great facility of ADSCs to differentiate with the application of an external mechanical stimulus to successfully create an in vitro reconstructed tendonlike structure with a microcapillary network. MATERIALS AND METHODS: Hyalonect meshes (Fidia Advanced Biopolymers, Abano Terme, Padova, Italy) were used as scaffold. Human ADSCs were seeded onto the biomaterials, and the cell/scaffold constructs were cultured under mechanical stress for up to 15 days. Human tenocytes were used in the same conditions as control. Performance was assessed by histology, immunochemistry, ultrastructure, and biomolecular analysis. RESULTS: Adipose-derived stem cells seeded onto Hyalonect adhered and differentiated along the entire surface of the biomaterial and began to infiltrate within its structure. Subsequently, endothelial cells migrated, forming a capillary in the new extracellular matrix. CONCLUSIONS: This technique allowed for the creation of a vascularized tendon equivalent that could easily be detached from the bioreactor, thus facilitating its implant at the lesion site. These results highlight the biologic performance of biodegradable hyaluronic acid-based (HYAFF-11) scaffolds, which were shown to be suitable for deposition of the autologous extracellular matrix critical for ADSCs differentiation.

In this study we report a preliminary investigation of the feasibility of non-woven/sponge fabrics of a hyaluronan derived biomaterials (benzyl ester of HA (HYAFF-11 FAB, Abano Terme, Italy) for the in vitro culture of rat hepatocytes and rat beta cells. Cell growth on hyaluronan derived biomaterials were tested in the presence of complete medium and in the presence of ECM (extracellular matrix) secreted by fibroblasts previously cultured into the scaffold. Hepatocytes and beta cells were extracted from rat liver/pancreas and seeded either on the HYAFF-11 scaffold alone, or on HYAFF-11 scaffold containing ECM. Direct assay of cell proliferation was performed with MTT test. For morphological observations samples were stained with hematoxylin and eosin. The results obtained by MTT test showed that hepatocytes cultivated in both the above described conditions were able to proliferate up to 14 days and Langerhans islet up to 21 days. After this time, cells started to undergo apoptosis. The morphological analyses showed cell aggregation in three-dimensional structures promoted by the fibers of the biomaterial. Our results confirmed that HYAFF-11 meshes represent a suitable scaffold for hepatocyte adhesion/Langerhans islet organization and proliferation. In particular, the presence of a fibroblast secreted extracellular matrix improves the biological property of the scaffold.

Hyaluronan (HL), a naturally occurring glycosaminoglycan, has been chemically modified through the esterification of its carboxylic groups with different types of alcohol. The physico-chemical properties of these new biopolymers allow the preparation of many biomaterials which may be used in several medical applications. In the present study both the biocompatibility and biodegradation of some water-insoluble HL esters have been evaluated, either as raw material or as manufactured devices after subcutaneous and intraperitoneal implantation in male rats. The inflammatory response and the degree of resorption for each tested material are reported. The relationships between the degree of esterification and the type of alcohol used with the above parameters are also investigated. © 1993.

The ideal bioartificial liver should be designed to reproduce as nearly as possible in vitro the habitat that hepatic cells find in vivo. In the present work, we investigated the in vitro perfusion condition with a view to improving the hepatic differentiation of pluripotent human liver stem cells (HLSCs) from adult liver. Tissue engineering strategies based on the cocultivation of HLSCs with hepatic stellate cells (ITO) and with several combinations of medium were applied to improve viability and differentiation. A mathematical model estimated the best flow rate for perfused cultures lasting up to 7 days. Morphological and functional assays were performed. Morphological analyses confirmed that a flow of perfusion medium (assured by the bioreactor system) enabled the in vitro organization of the cells into liver clusters even in the deeper levels of the sponge. Our results showed that, when cocultured with ITO using stem cell medium, HLSCs synthesized a large amount of albumin and the MTT test confirmed an improvement in cell proliferation. In conclusion, this study shows that our in vitro cell conditions promote the formation of clusters of HLSCs and enhance the functional differentiation into a mature hepatic population.

The aim of this study was to develop a prosthetic graft that could perform as a small-diameter vascular conduit for vein regeneration. The difficulty of obtaining significant long-term patency and good wall mechanical strength in vivo has been a significant obstacle in achieving small-diameter vein prostheses. Fifteen Male Wistar rats weighing 250-350 g were used. Tubular structures of hyaluronan (HYAFF-11 tubules, 2 mm diameter, and 1.5 cm length) were implanted in the vena cava of rats as temporary absorbable guides to promote regeneration of veins. Performance was assessed at 30, 60, and 90 days after surgery by histology (hematoxylin-eosin and Weighert solution) and immunohistochemistry (antibodies to von Willebrand factor and to Myosin Light-Chain Kinase). These experiments resulted in two novel findings: (1) sequential regeneration of vascular components led to complete vein wall regeneration 30 days after surgery; (2) the biomaterial used created the ideal environment for the delicate regeneration process during the critical initial phases, yet its biodegradability allowed for complete degradation of the construct 4 months after implantation, at which time, a new vein remained to connect the vein stumps. This work demonstrates the complete vena cava regeneration inside the hyaluronic acid-based prosthesis, opening new perspective of microsurgical applications, like replantation of the upper limb, elongation of vascular pedicle of free flaps, cardiovascular surgery, and pediatric microvascular surgery.

‘Small is beautiful’ – this should be the slogan of nanoscientists. Indeed, working with particles less than 100 nm in size, nanotechnology is on the verge of providing a host of new materials and approaches, revolutionizing applied medicine. The obvious potential of nanotechnology has attracted considerable investment from governments and industry hoping to drive its economic development. Several areas of medical care already benefit from the advantages that nanotechnology provides and its application in wound healing will be reviewed in this article.

OBJECTIVE: In recent years, research on stem cells has been focused on the development of personalized cell-based therapies. Owing to their homing properties, adult human stem cells are a promising source of autologous cells to be used as therapeutic vehicles. Multiple potential sources for clinically useful stem and progenitor cells have been identified, including autologous and allogenic embryonic, fetal and adult somatic cells from neural, adipose and mesenchymal tissue. In the present report, we describe a simple protocol to obtain an enriched culture of adult stem cells organized in neurospheres from two post-natal tissues: skin and adipose tissue. METHODS: Adult stem cells isolated from skin and adipose tissue derived from the same adult donor were amplified under varying conditions related to the coating of the chamber slide and the presence of serum and/or growth factors, such as with EGF and FGF2. Neurospheres were then expanded and evaluated in terms of proliferation and gene expression. RESULTS: Adipose and skin derived neurospheres were comparable in size, quantity of cells and genes expressed. Cells from both types of tissue grew optimally without slide coating, in the presence of serum and with the combined addition of FGF2 and EGF. DISCUSSION: We describe a method for isolating and improving a population of multipotent adult precursor cells from the two most accessible adult tissue sources: skin and adipose tissue. This autologous adult stem cell population could be used for cell replacement or cell therapies.

Tissue engineering is a multidisciplinary field focused on in vitro reconstruction of mammalian tissues. In order to allow a similar three-dimensional organization of in vitro cultured cells, biocompatible scaffolds are needed. This need has provided immense momentum for research on “smart scaffolds” for use in cell culture. One of the most promising materials for tissue engineering and regenerative medicine is a hyaluronan derivative: a benzyl ester of hyaluronan (HYAFF®). HYAFF® can be processed to obtain several types of devices such as tubes, membranes, non-woven fabrics, gauzes, and sponges. All these scaffolds are highly biocompatible. In the human body they do not elicit any adverse reactions and are resorbed by the host tissues. Human hepatocytes, dermal fibroblasts and keratinocytes, chondrocytes, Schwann cells, bone marrow derived mesenchymal stem cells and adipose tissue derived mesenchymal stem cells have been successfully cultured in these meshes. The same scaffolds, in tube meshes, has been applied for vascular tissue engineering that has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue.

Hydrogels are water-swollen polymeric materials that maintain a distinct three-dimensional structure. They were the first biomaterials designed for use in the human body. Traditional methods of biomaterial synthesis include crosslinking copolymerization, crosslinking of reactive polymer precursors, and crosslinking via polymer-polymer reaction. These methods of hydrogel synthesis were limited in the control of their detailed structure. Other inadequacies of traditional hydrogels have been poor mechanical properties and slow or delayed response times to external stimuli. The huge field of biomaterials research has received a strong revitalization by several novel approaches in hydrogel design. Enhanced biomechanical properties of hydrogel preparation, superporous and comb-type grafted hydrogels with fast response times, and self-assembly are just a few examples of hydrogel biomaterials with a smart future. Potential applications of all the types of hydrogels include: tissue engineering, synthetic extracellular matrix, implantable devices, biosensors, separation systems (valves to control permeability across porous membranes, or materials for affinity separation based on the specific recognition of monomelic strands), materials controlling the activity of enzymes, phospholipid bilayer destabilizing agents, materials controlling reversible cell attachment, nanoreactors with precisely placed reactive groups in three-dimensional space and smart microfluidics. With regard to the applications of hydrogels in recent years, particular attention has been devoted to drug delivery, clinical application as well as to the use of hydrogels as scaffolds for tissue engineering and regenerative medicine. Among the materials used for regenerative applications, hydrogels seem very promising and are receiving increasing attention due to their ability to entrap large amount of water, good biocompatibility and the ability to mimic tissue environment.

Vascular tissue engineering has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue. The missing link in tissue-engineered blood vessels is elastin biosynthesis. Several biomaterials are currently used but few support elastin biosynthesis in a 3-D array. In previous studies, we demonstrated that a hyaluronan-based scaffold (HYAFF-11) grafted in the infrarenal rat aorta successfully guided the complete regeneration of a well-functioning small-diameter (2 mm) neoartery. The aim of the present study was to test the ability of HYAFF-11 biodegradable grafts to develop into neovessels of larger size (4 mm) in a porcine model, focusing on extracellular matrix (ECM) remodeling and elastin biosynthesis. HYAFF-11 tubes (diameter 4 mm, length 5 cm) were implanted in an end-to-end fashion in the common carotid artery. Grafts were analyzed for patency with a Duplex scan every 15 days. ECM components were evaluated by histological and molecular biological methods. All the animals survived the observation period without complications. Intimal hyperplasia (initiating at the anastomotic site) and graft thrombosis led to 3 cases of partial or complete occlusion, as demonstrated by histological examination. There were no signs of stenoses or aneurysms in the remaining grafts. After 5 months, the biomaterial was almost completely degraded and replaced by a neoartery segment composed of mature smooth muscle cells, collagen, and elastin fibers organized in layers and was completely covered on the luminal surface by endothelial cells (vWF(+)). Whereas in previous small animal studies, patency rates were not optimal, those obtained in the present study using hyaluronan-based grafts of larger size confirmed the ability of these constructs to guide the development of a well-functioning neoartery, with the remarkable additional attribute of facilitating the formation of organized layers of elastin fibers.

The regeneration of damaged organs requires that engineered tissues mature when implanted at sites of injury or disease. We have used new analytic techniques to determine the extent of tissue regeneration after treatment of knee injury patients with a novel cartilage tissue engineering therapy and the effect of pre-existing osteoarthritis on the regeneration process. We treated 23 patients, with a mean age of 35.6 years, presenting with knee articular cartilage defects 1.5 cm2 to 11.25 cm2 (mean, 5.0 cm2) in area. Nine of the patients had X-ray evidence of osteoarthritis. Chondrocytes were isolated from healthy cartilage removed at arthroscopy. The cells were cultured for 14 days, seeded onto esterified hyaluronic acid scaffolds (Hyalograft C), and grown for a further 14 days before implantation. A second-look biopsy was taken from each patient after 6 to 30 months (mean, 16 months). After standard histological analysis, uncut tissue was further analyzed using a newly developed biochemical protocol involving digestion with trypsin and specific, quantitative assays for type II collagen, type I collagen, and proteoglycan, as well as mature and immature collagen crosslinks. Cartilage regeneration was observed as early as 11 months after implantation and in 10 out of 23 patients. Tissue regeneration was found even when implants were placed in joints that had already progressed to osteoarthrosis. Cartilage injuries can be effectively repaired using tissue engineering, and osteoarthritis does not inhibit the regeneration process.

The difficulty of obtaining significant long-term patency and good wall mechanical strength in vivo has been a significant obstacle in achieving small-diameter vascular prostheses. The aim of the present study was to develop a prosthetic graft that could perform as a small-diameter vascular conduit. Tubular structures of hyaluronan (HYAFF-11 tubules, 2 mm diameter, 1 cm length) were grafted in the abdominal aorta of 30 rats as temporary absorbable guides to promote regeneration of vascular structures. Performance was assessed by histology, immunohistochemistry, and ultra-structural analysis. These experiments resulted in three novel findings: 1) complete endothelialization of the tube's luminal surface occurred; 2) sequential regeneration of vascular components led to complete vascular wall regeneration 15 days after surgery; and 3) the biomaterial used created the ideal environment for the delicate regeneration process during the critical initial phases, yet its biodegradability allowed for complete degradation of the construct four months after implantation, at which time, a new artery remained to connect the artery stumps. This study assesses the feasibility to create a completely biodegradable vascular regeneration guide in vivo, able to sequentially orchestrate vascular regeneration events needed for very small artery reconstruction.

Successful in vitro reconstruction of skin requires the inclusion of several cell types that give rise in coculture to the specific elements present in native skin, and the appropriate scaffolding structure to house and support these cells. In addition to the two main structural components, epidermis and dermis, one critical apparatus of the skin is a capillary network that guarantees adequate perfusion of nutrients and oxygen. The aim of the present study was to develop an in vitro coculture system that assumed the human dermal-epidermal architecture and included a microcapillary network in a three-dimensional biomaterial that guaranteed ease of handling in a clinical setting. Endothelialized skin (ES) was prepared by coculturing three human cell types: keratinocytes, fibroblasts, and endothelial cells, obtained from human full-thickness skin samples, in scaffolds produced from modified hyaluronic acid. Results were evaluated by histological and immunohistochemical analyses at different time points. In vitro, engineered skin obtained with this composite culture developed into a well-differentiated upper layer of stratified keratinocytes lining a dermal-like structure, in which fibroblasts, extracellular matrix and a microvascular network were present. Furthermore, the biodegradable fabric produced from hyaluronic acid and used as the scaffolding support for this in vitro constructed skin graft greatly facilitated handling in the perioperative period.

PURPOSE. Plasminogen activators play a role, not only in fibrinolysis but also in events such as chemotaxis, collagen degradation, and cell spreading. The serine protease urokinase (uPA) is a potent chemoattractant for leukocytes that may be involved in the pathogenesis of severe forms of allergic conjunctivitis such as vernal keratoconjunctivitis (VKC). METHODS. Tear and peripheral blood samples were obtained from 20 patients with active VKC and from 19 normal subjects who formed the control group. Levels of plasminogen activity, uPA, tissue plasminogen activator (tPA), and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1) were measured in tears and plasma of patients with VKC. The presence of tPA, uPA, and urokinase receptor (uPAR) in conjunctival tissues were evaluated by immunohistochemistry. uPA, uPAR, and PAI-1 expression and production were measured in conjunctival epithelial cell and fibroblast cultures treated with cytokines. RESULTS. Tear levels of uPA and tPA and tear plasminogen activity levels were significantly greater in patients with VKC than in control subjects. Increased staining for uPA and uPAR was found in VKC tissues compared with normal conjunctiva. Both conjunctival epithelial cells and fibroblasts demonstrated an increased expression of uPAR after exposure to IL-4 or -13, whereas uPA was highly expressed by epithelial cells exposed to IL-4. PAI-1 levels in culture medium were increased in IL-4-exposed epithelial cells compared to nonstimulated cells and were decreased in fibroblast culture. CONCLUSIONS. Increased expression of fibrinolytic system components and imbalance between plasminogen activators and PAI may be involved in the pathogenesis of severe allergic conjunctivitis, thus contributing to inflammatory cell migration and tissue remodeling. (Invest Ophthalmol Vis Sci. 2005;46: 1364‐1370) DOI:10.1167/iovs.04-1196

Generation of a vascular network is a hallmark of solid tumor growth, and attempts to switch off the tumor angiogenic phenotype are promising. However, this angiogenic potential might also be exploited to obtain incorporation into tumor vessels of genetically modified third-party cells, which could behave as targets of immunologic or pharmacologic attack. With this in mind, we addressed the efficiency and selectivity of third-party cell recruitment into experimental tumors generated in severe combined immunodeficiency mice. The animals were inoculated intraperitoneally with human ovarian carcinoma cell lines and with beta-galactosidase (beta-gal)-transduced human umbilical vein endothelial cell (HUVEC) or human fibroblasts. Transgenic HUVEC were scattered in tumors, but not in normal mouse tissues; immunohistochemical analysis revealed their selective homing to tumor vascular structures, over 50% of which contained beta-gal(+) cells. Injection of beta-gal-transduced human fibroblasts was also associated with transgenic cell incorporation into tumor masses; however, beta-gal(+) fibroblasts did not home to tumor blood vessels and were only localized within the tumor stroma. These findings show that the recruitment of primary third-party cells into the different compartments of experimentally induced tumors is an efficient and selective phenomenon and indicate possible alternative ways of confronting the tumor angiogenic potential in cancer therapy.

Objective: To identify the CD44-receptor-mediated effects of 5–7×105MW hyaluronan (HA, Hyalgan®) on cell viability in normal and damaged human chondrocyte primary cultures isolated from articular cartilage.Design: Primary cultures of human chondrocytes were established from normal articular biopsies and expanded to the second culture passage. The dose–response effects of HA on the viability of normal cultures were identified. Chondrocytes were then treated with either hypoxanthine (2mM) and xanthine oxidase (20–60mU), or with activated polymorphonuclear leukocytes (PMNs) to induce injury. Damaged and control cells were then treated with 5–7×105HA in the previously identified optimal dose of 0.05mg/ml. Viability was assessed at specific time periods for the chemically and PMN-damaged cells. To identify if HA effects were mediated by the CD44 receptor, chondrocytes were incubated with anti-CD44 antibody at saturating concentrations (5μg/ml for 100000 cells) to produce a maximum inhibition of HA binding. Cells were evaluated using the MTT viability assay, histology, electron microscopy and immunohistochemistry.Results: Direct addition of HA (optimal dose, 0.5mg/ml) significantly increased cell survival in normal chondrocyte primary cultures (P

PURPOSE. To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling. METHODS. Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)- and tumor necrosis factor (TNF)-. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR. RESULTS. IL-4 and -13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN- elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and -13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF- increased production of MMP-1 and -9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines. CONCLUSIONS. IL-4 and -13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN- and TNF- .( Invest Ophthalmol Vis Sci. 2003;44:183‐189) DOI

The tolerability and safety of hyaluronan-based three-dimensional scaffolds as a culture vehicle for mesenchymal progenitor cells was investigated in this pilot study. The proliferation patterns and extracellular matrix production of rabbit and human mesenchymal, bone-marrow-derived progenitors first were characterized in vitro. Subsequently rabbit autologous cells were cultured in this hyaluronan-based scaffold and implanted in a full-thickness osteochondral lesion. In vitro histologic findings showed that mesenchymal progenitor cells adhered and proliferated onto the hyaluronan-derived scaffold. Human stem cells were shown to produce the main extracellular matrix molecules, accompanied by an occasional synthesis of mature type II collagen. In vivo data demonstrated that the biomaterial, with or without mesenchymal progenitors, did not elicit any inflammatory response and was completely degraded within 4 months after implantation. With regard to the efficacy of this cell therapy, even among the small number of animals tested there was histologic evidence that lesions filled with the biomaterial, either seeded or unseeded with cells, achieved a faster and better healing compared to empty controls. The present data suggest that the hyaluronan-based scaffolds are well tolerated and safe and may be a valuable delivery vehicle for tissue engineering in the repair of articular cartilage defects.

The current study was designed to investigate the biological role of small extracellular matrix fragments in wound healing. Human burn eschar tissue was digested with bacterial collagenase, and small aminoacidic fragments were inoculated in both human dermal fibroblast cultures and polyvinyl alcohol sponges implanted subcutaneously in the rat. Proliferation assays on cell cultures and biochemical and histologic analyses of the animal model were then performed, Results showed that fibroblasts treated with low concentrations of eschar fragments duplicated significantly faster than controls. Biochemical and histologic data from sponge implants showed that the inflammatory response was augmented by eschar-derived fragments at postoperative day 2, whereas protein and hydroxyproline synthesis were decreased at day 14. In conclusion, these data substantiate that the application of bacterial collagenase to debride necrotic tissue may have an indirect healing effect resulting from the local release of bioactive matrix-derived fragments.

Articular cartilage has a very limited self-repairing capacity; thus, chondral lesions normally result in chronic degeneration and, eventually, osteoarthritis development. Currently, tissue engineering offers a new tool for the clinical treatment of osteochondral defects. The present investigation aimed to develop an in vitro engineered cartilage using a new class of semisynthetic scaffolds. Two nonwoven meshes of hyaluronan esters (Hyaff(R) derivatives) were seeded with sternal chick embryo chondrocytes cultured for up to 21 days, after which time they were assessed for both the cellular growth profile and histological features. Avian chondrocytes easily adhered and proliferated onto hyaluronan-based scaffolds, demonstrating a significant preference for the fully esterified benzylic form. Histochemical staining revealed the presence of a neosynthesized glycosaminoglycan-rich extracellular matrix, and immunohistochemistry confirmed the deposition of collagen type II. Moreover, ultrastructural observations supported evidence that chondrocytes grown onto a hyaluronan-derived three-dimensional scaffold maintained their unique phenotype and organization in a cartilage-like extracellular matrix. These findings support the further pursuit of a transplantable engineered cartilage using human chondrocytes for the regeneration of chondral lesions.

Objective: To compare hyaluronan and its α -methyl prednisolone derivative(HYC-141) with respect to their potential to directly cause tissue damage and to protect tissues from attack by reactive oxygen species. Methods: Cartilage samples and chondrocytes were isolated from 15-day chickembryos and were exposed to enzyme-based and activated inflammatory cell-free radical generating systems in the presence of varying concentrations of α -methyl prednisolone, hyaluronan, and HYC-141. Tissue and cell vitality was measured by determining the incorporation of radioactive sulphate into newly synthesized glycosaminoglycans. Results: Only α -methyl prednisolone caused a significant decrease in biosyntheticactivity. All the tested substances were capable, to some extent, of protecting tissues and cells from damage by reactive oxygen species; HYC-141 demonstrated the greatest protective effect. Conclusion: These data suggest that HYC-141 may possess certain advantages over the individual component molecules in the local treatment of arthropathies.

During wound healing, cells are attracted by many stimuli to the injured area. Many substances are known to have a chemotactic effect on different cells. Among those, human collagen breakdown products (HCBP) have been shown to be chemotactic in vitro for fibroblasts and white blood cells. In our experiments, collagen (types I and III) extracted from the human placenta was enzymatically digested with bacterial collagenase (Knoll AG, Germany). Breakdown peptides were then used both in vitro and in vivo to evaluate their proliferative and chemotactic activities. HCBP were demonstrated to have a proliferative effect on murine and human fibroblasts, while they showed a chemotactic effect in a model of wound healing in the rat.

All wounds involve connective tissue and heal by the process called repair. The events occurring in the repair process are such that the original connective tissue architecture is lost. The new tissue, called repair tissue, restores continuity within the wound space and consists first of granulation tissue and later scar tissue. The latter shows several compositional and architectural differences from the tissue native to the site. In all of the repair phases, the connective tissue reaction is the principal event. Thus knowledge of connective tissue biology is essential to understanding the repair process.

The biocompatibility of semisynthetic polymers formed from hyaluronic acid esters has been studied using fibroblast cultures. The polymers, added to the culture medium, used either in powdered form or as thin membranes, behave as inert materials. The cells used in the experiments grow normally in the culture dishes. With regard to adhesiveness the cells were not able to spread on the biomembranes and tended to form isolated clusters of round cells. Human fibronectin, placental collagen (type I-IV) and fibrin could be stratified on biomembranes. When these molecules reacted with the biomaterial the film became suitable for fibroblasts spreading and growth.

15-day-chick-embryo fibroblasts and chondroblasts were cultured in the presence of high and low molecular weight exogenous hyaluronic acid (HA). Growth range and incorporation of radio-labelled sulphate and proline were determined. HA reduced cell proliferation to about 75% of controls, while incorporation of radiolabelled sulphate and proline was higher in HA-treated cultures of both chondroblasts and fibroblasts. The effect was not due to the polyanionic or polymeric nature of the molecule and appeared to be highly specific for HA.

Histological and chemical studies were carried out on the elastin and collagen content of the normal and the obstructed urinary bladder wall. The observations were made on 3 different age groups: (1) 3-month-old children with vesical outlet obstruction, (2) children aged between 4 and 8 years with partial vesical obstruction, and (3) adults with a chronically obstructed bladder. An increase in elastic tissue was shown only in the newborn and in the adults. The collagen did not vary in any of the cases studied. The elastin showed an increase in polar amino acids in the pathological organs compared with the normal. A similar increase in elastin was observed in normal individuals as a result of the ageing process.

Using an SE-Sephadex column, it was possible to separate five peptide fractions from α-elastin. Each fraction has been separately analyzed in order to determine the amino acid composition, the N-terminal residues and the homogeneity on Agarose gel and by disc electrophoresis. The amino acid composition of the single fractions reveals no significant differences when compared with native elastin. Serine, glutamic acid, glycine and alanine are the only N-terminal residues present. The absence of other N-terminal residues has been interpreted as due to a selective cleaving by oxalic acid which probably occurs only at specific amino acids. As far as the homogeneity of the single fractions is concerned, the data obtained with agarose gel and by disc electrophoresis, indicate that these fractions are not pure but mixtures of microdispersed peptides.

Using chemical and histochemical techniques, it has been possible to calculate the exact amount and distribution of the elastin in normal and abnormal ureters in children. From the quantitative analyses, it has been calculated that in the dilated ureters the elastin content represents the 3% to 5% of the organic material, while in the normal ureters it is less than 1%.

The calcified matrix of the hen eggshell has been demineralized with the EDTA. Aliquots of this material are soluble in water and have been characterized by column chromatography and by chemical analyses. Of particular interest is the high hexosamine and uronic acid content, which confirms the protein-polysaccharide nature of this water-soluble material. The calcium ion binding to the eggshell matrix has been studied by the equilibrium dialysis technique at different pH values, with both free and blocked carboxylic groups. The material with the free carboxylic side chain groups binds more calcium ions with increasing pH value. When the carboxylic groups have been previously blocked with a water-soluble carbodiimide, the calcium ion binding rapidly decreases. The residual capacity to bind calcium ions in the material with the carboxylic functions modified is probably due to the sulfate ions. In agreement with previous observations on other calcified substrates, the calcium ion binding seems to depend on the presence of ionized carboxylic functions of the matrix.

The binding of Ca2+ to the soluble products obtained from elastin has been determined by titration using murexide as an indicator of free Ca2+. The soluble peptides were represented by the α-elastin in toto and by some single fractions obtained by SE-Sephadex column chromatography of the α-elastin. Ca2+ binding was shown to be dependent on pH and Ca2+ concentration and increases with increasing pH. The data support the possibility that the electrostatic interaction occurs between the ionized carboxylic groups of the elastin peptides and Ca2+. This is confirmed by the evidence that the peptides in which the carboxylic groups were reacted with the glycine ethyl ester and water-soluble carbodiimide, did not bind Ca2+.