A stereotypic,transplantable liver tissue-culture system

A method of coculturing adult rat hepatic parenchymal cells (PC) and stromal cells in a three-dimensional framework of nylon filtration screens or biodegradable polymer meshes was developed in our laboratory. Rat liver stroma, which includes vascular and bile duct endothelial cells, fat-storing cells, fibroblasts, and Kupffer cells, were isolated by gradient centrifugation afterin situ liver perfusion and expanded in monolayer culture prior to seeding onto nylon screens or bioresorbable polyglycolic acid (PGA) polymers oriented into a felt-like construct. A second inoculum of freshly isolated PC was applied after the stromal cells became established. Histological analyses revealed that PC proliferation occurred until all available space for expansion within the template was exhausted. These cells retained their rounded morphology, and after 4–5 wk 7–9 “layers” of PC filled the 140-μm deep template. Dioxin-inducible cytochrome P450 activity was detected for up to 58 d in culture, and albumin, fibrinogen, transferrin, and soluble fibronectin were detected in the medium by enzyme-linked immunosorbent assay (ELISA) for 48 d in vitro. Immunohistochemical analysis of sections through the cultures confirmed the presence of these proteins as well as cytokeratin at the cellular level; the extracellular matrix stained for both collagen type III and laminin. Long-term PC proliferation and function were enhanced by the presence of stromal cells as well as by a meshwork template whose geometry allows the interaction of PC with stroma and matrix on several different planes. To permit transplantation, co-cultures of hepatic PC and stromal cells were established on PGA felt constructs instead of nylon screens. After 24 d in vitro, these constructs were grafted into sites in the mesentery, omentum, and sub-cutaneous tissues of adult Long-Evans rats. The growth of hepatocytes after 30 din situ was evident by histological analysis; grafts of co-cultures regenerated a liver-like architecture consisting of sinusoids and putative biliary structures. In addition, PC at these extrahepatic graft sites were positive for albumin, transferrin, and fibrinogen synthesis by immunohistochemistry. Graft survival was enhanced when recipients were subjected to 40% hepatectomy. Hepatic PC:stromal cell cocultures may prove useful in the restoration of liver function either by direct transplantation using PGA or similar templates, or as extracorporeal devices, using nylon screens.     

Methylated and pegylated PLA–PCL–PLA block copolymers via the chemical modification of di‐hydroxy PCL combined with the ring opening polymerization of lactide

Methylated and pegylated poly(lactide)‐block‐poly(ε‐caprolactone)‐block‐poly(lactide) copolymers, PLA–P(CL‐co‐CLCH₃)–PLA and PLA–P(CL‐co‐CLPEG)–PLA, were prepared in three steps: combining the formation of carbanion‐bearing dihydroxylated‐PCL, the coupling of iodomethane or bromoacetylated α‐hydroxyl‐ω‐methoxy‐poly(ethylene glycol) onto the carbanionic PCL, and finally the ring opening polymerization of DL ‐lactide initiated by the preformed grafted diOH‐PCL copolymers. The resulting block copolymers exhibited lower crystallinity, melting temperature, and hydrophobicity with respect to the original PCL. Degradation of the grafted copolymers was investigated in the presence of Pseudomonas cepacia lipase and compared with that of the triblock copolymer precursor. It is shown that the presence of the grafted substituents affected the enzymatic degradation of PCL segments. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4196–4205, 2005     

In Vivo Justification of Using Endobiliary Stents Made of Polyhydroxyalkanoates

Treatment of patients with mechanical jaundice (MJ) has been one of the topical problems of current medicine. The fraction of patients having MJ of oncologic character that can undergo radical surgery does not exceed 25–30%. A way out of such situation is to use minimally invasive endobiliary interventions, the main type of which is endoprosthesis replacement of bile duct lumens. The aim of the work was to study the biological properties of the experimental models of polymer stents made of PHA for endobiliary prosthetics and to investigate the biological properties of PHA suture material for forming of biliary-enteric anastomoses (cholecystoduodenostomy). Experiments were performed on 20 adult mongrel dogs, weighing 10–12 kg. The animals were divided to three groups: the negative control group (intact animals); the positive control group (the animals with implanted endobiliary silicon stents); and the experimental group (the animals with the PHA stents). The animals were monitored for 100 days. The clinical blood analysis was made before the operation, on day 7, 30, 60 and 100 days. During autopsy the presence of exudate, commissural process in the free abdominal cavity and subhepatic spatium, the appearance of choledoch where the prosthesis was located, as well as the appearance of cholecystoenterostomy, liver and duodenum. We did not found any signs of inflammation, cicatrical changes were estimated in the free abdominal cavity and subhepatic spatium. All implanted PHA stents were at their initial places of implantation. After the end of the experiment inflammatory reaction and anastomositis were absent. Macroscopic changes of liver and duodenum were also not detected. Liver function did not have any pathological deviations. These positive results give grounds to conclude that application of PHA as endobiliary stents in reconstructive surgery of bile passages and as suture material is a promising technology.     

A Polymer for Application as a Matrix Phase in a Concept of In Situ Curable Bioresorbable Bioactive Load-Bearing Continuous Fiber Reinforced Composite Fracture Fixation Plates

The use of bioresorbable fracture fixation plates made of aliphatic polyesters have good potential due to good biocompatibility, reduced risk of stress-shielding, and eliminated need for plate removal. However, polyesters are ductile, and their handling properties are limited. We suggested an alternative, PLAMA (PolyLActide functionalized with diMethAcrylate), for the use as the matrix phase for the novel concept of the in situ curable bioresorbable load-bearing composite plate to reduce the limitations of conventional polyesters. The purpose was to obtain a preliminary understanding of the chemical and physical properties and the biological safety of PLAMA from the prospective of the novel concept. Modifications with different molecular masses (PLAMA-500 and PLAMA-1000) were synthesized. The efficiency of curing was assessed by the degree of convergence (DC). The mechanical properties were obtained by tensile test and thermomechanical analysis. The bioresorbability was investigated by immersion in simulated body fluid. The biocompatibility was studied in cell morphology and viability tests. PLAMA-500 showed better DC and mechanical properties, and slower bioresorbability than PLAMA-1000. Both did not prevent proliferation and normal morphological development of cells. We concluded that PLAMA-500 has potential for the use as the matrix material for bioresorbable load-bearing composite fracture fixation plates.     

Enlarging the library of poly‐(L‐lysine citramide) polyelectrolytic drug carriers

Poly‐(L ‐lysine citramide) is a degradable drug carrier of the polyelectrolyte type that is composed of citric acid and L ‐lysine building blocks. In a previous work, poly‐(L ‐lysine citramide) was synthesized by the interfacial polycondensation of α‐hydroxy acid protected citryl dichloride with COOH‐protected lysine diamine. Because of head‐to‐head and head‐to‐tail and tail‐to‐tail linkages in the chains as well as various side reactions such as deprotection of the α‐hydroxy acid moieties and intramolecular imide ring formation, a very large family of degradable polyelectrolyte copolymers was obtained. All the members of this family hydrolytically degrade to the same end products. In this study, another route was explored based on the polycondensation of α‐hydroxy acid protected citric acid pentafluorophenyl diesters, namely, citrobenzal dipentafluorophenyl and citrochloral dipentafluorophenyl with N‐N′‐trimethylsilylated COOH‐protected L ‐lysine. The resulting polymers were characterized by IR, NMR, and size exclusion chromatographic analyses. The resulting chain structures and repeat units were identified from these characterizations and are discussed as compared with characteristics exhibited by analogous polymers resulting from interfacial polycondensation. Differences observed at the intermediate stage involving protected polymers were largely erased during the final deprotection stage because of imide formation during final hydrolysis under the selected conditions. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 3475–3484, 2001     

Clinical Application of Resorbable Polymers in Guided Bone Regeneration

Summary : Guided bone regeneration was shown to be successful in vitro and in vivo using resorbable or nonresorbable materials. Resorbable material has the advantage of progressive substitution by bone. Resorbable polymers of ∝-hydroxy acids like polylactide or polyglycolide are commonly used for tissue engineering and in guided bone regeneration. In clinical studies, guided bone regeneration was successful in non-weight bearing bone, e.g. in dental surgery and craniofacial surgery. This paper reports the preliminary result of using resorbable poly(L/DL-lactide) 80/20% scaffolds in weight bearing bone with infected large segmental defects as well as in small bony defects of hand due to benign tumour, bone graft donor sites and as an adjunct for joint fusion. Resorbable polylactide implants were used in the form of membranes, large 3-D sponges, chips or as injectable paste. Implants were impregnated with marrow blood to add an osteoinductive component. Long-term follow up revealed that these implants are promising candidates for bone graft substitutes.     

Development of a Polymer‐Based Biodegradable Neurovascular Stent Prototype: A Preliminary In Vitro and In Vivo Study

Biodegradable stents are not established in neurovascular interventions. In this study, mechanical, radiological, and histological characteristics of a stent prototype developed for neurovascular use are presented. The elasticity and brittleness of PLA 96/4, PLDL 70/30, PCL, and PLGA 85/15 and 10/90 polymers in in vitro experiments are first analyzed. After excluding the inapt polymers, degradability and mechanical characteristics of 78 PLGA 85/15 and PLGA 10/90 stent prototypes are analyzed. After excluding PLGA 10/90 stents because of rapid loss of mass PLGA 85/15 stents in porcine in vivo experiments are analyzed. Angiographic occlusion rates 7 d, 1 month, 3 months, and 6 months after stent implantation are assessed. Histological outcome measures are the presence of signs of inflammation, endothelialization, and the homogeneity of degradation after six months. One case of stent occlusion occurs within the first 7 d. There is a prominent foreign‐body reaction with considerable mononuclear and minor granulocytic inflammation combined with incomplete fragmental degradation of the struts. It is possible to produce a stent prototype with dimensions that fit the typical size of carotid arteries. Major improvements concerning thrombogenicity, degradation, and inflammatory response are required to produce biodegradable stents that are suitable for neurovascular interventions.     

Synthesis and Characterization of Fumarate Copolyesters for Use in Bioresorbable Bone Cement Compositions

Unsaturated amorphous copolyesters of varied composition were prepared by transesterification copolymerization of diethyl fumarate, and two diols, 1,2‐propanediol and 2‐methyl‐1,3‐propanediol. The copolyesters were characterized by IR, ¹H‐ and ¹³C‐NMR, GPC, DSC, and TGA. The glass transition is changing with composition from 0°C to 19°C as the content of 1,2‐propanediol residue in the copolyester increases. The copolyester structure and composition have an impact on the compressive strength and hydrolytic stability of the composites prepared by crosslinking the fumarate double bonds with N‐vinyl pyrrolidone in the presence of inorganic filler, calcium sulfate dihydrate, with the addition of a radical initiator, benzoyl peroxide, at ambient temperatures.     

A Novel Resorbable Composite Material Containing Poly(ester-co-urethane) and Precipitated Calcium Carbonate Spherulites for Bone Augmentation—Development and Preclinical Pilot Trials

Polyurethanes have the potential to impart cell-relevant properties like excellent biocompatibility, high and interconnecting porosity and controlled degradability into biomaterials in a relatively simple way. In this context, a biodegradable composite material made of an isocyanate-terminated co-oligoester prepolymer and precipitated calcium carbonated spherulites (up to 60% w/w) was synthesized and investigated with regard to an application as bone substitute in dental and orthodontic application. After foaming the composite material, a predominantly interconnecting porous structure is obtained, which can be easily machined. The compressive strength of the foamed composites increases with raising calcium carbonate content and decreasing calcium carbonate particle size. When stored in an aqueous medium, there is a decrease in pressure stability of the composite, but this decrease is smaller the higher the proportion of the calcium carbonate component is. In vitro cytocompatibility studies of the foamed composites on MC3T3-E1 pre-osteoblasts revealed an excellent cytocompatibility. The in vitro degradation behaviour of foamed composite is characterised by a continuous loss of mass, which is slower with higher calcium carbonate contents. In a first pre-clinical pilot trial the foamed composite bone substitute material (fcm) was successfully evaluated in a model of vertical augmentation in an established animal model on the calvaria and on the lateral mandible of pigs.     

Synthesis and rheological properties of polylactide/poly(ethylene glycol) multiblock copolymers

Ring-opening polymerization of D,L-lactide was carried out in the presence of poly(ethylene glycol), using Zn powder as catalyst. The hydroxyl-capped PLA-PEG-PLA triblock copolymers were coupled with adipoyl chloride at different molar ratios under mild conditions. N-Dimethylaminopyridine (DMAP) was used as catalyst of the coupling reaction. The resulting PLA/PEG multiblock copolymers were characterized by various analytical techniques such as IR, 1H NMR, SEC, and DSC. Sol-gel transition properties of the multiblock copolymers were investigated by mechanical rheology. The data showed that the sol-gel transition temperature and the transition modulus increased with increasing molecular weight and the solution concentration of the multiblock copolymers. [Graph: see text] Variation of storage modulus (G') and loss modulus (G') as a function of temperature for a 20% sample of MB3.