Human clinical experience with adipose precursor cells seeded on hyaluronic acid-based spongy scaffolds.

Histioconductive approaches to soft-tissue defects use scaffolds seeded with lineage- and tissue-specific progenitors to generate tissue which should reside in equilibrium with adjacent tissue. Scaffolds guide histiogenesis by ensuring cell-cell and cell-matrix interactions. Hyaluronic acid-based (HA) preadipocyte-seeded scaffolds were evaluated for their adipo-conductive potential and efficacy in humans. Preadipocytes were isolated from lipoaspirate material and seeded on HA scaffolds. The cellular bio-hybrid (ADIPOGRAFT) and an acellular control scaffold (HYAFF11) were implanted subcutaneously. At specific time points (2, 8 and 16 weeks) explants were analyzed histopathologically with immunohistochemistry. No adverse tissue effects occurred. Volume loss and consistent degradation of the HYAFF11 scaffolds compared to the ADIPOGRAFT group indicated progressive tissue integration. No consistent histological differences between both groups were observed. By 8 weeks all void spaces within the scaffolds were filled with cells with pronounced matrix deposition in the ADIPOGRAFT bio-hybrids. Here we show that HA scaffolds were stable cell carriers and had the potential to generate volume-retaining tissue. However, no adipogenic differentiation was observed within the preadipocyte-seeded scaffolds.

Differential cartilaginous tissue formation by human synovial membrane, fat pad, meniscus cells and articular chondrocytes.

OBJECTIVE: To identify an appropriate cell source for the generation of meniscus substitutes, among those which would be available by arthroscopy of injured knee joints. METHODS: Human inner meniscus cells, fat pad cells (FPC), synovial membrane cells (SMC) and articular chondrocytes (AC) were expanded with or without specific growth factors (Transforming growth factor-beta1, Fibroblast growth factor-2 and Platelet-derived growth factor bb, TFP) and then induced to form three-dimensional cartilaginous tissues in pellet cultures, or using a hyaluronan-based scaffold (Hyaff-11), in culture or in nude mice. Human native menisci were assessed as reference. RESULTS: Cell expansion with TFP enhanced glycosaminoglycan (GAG) deposition by all cell types (up to 4.1-fold) and messenger RNA expression of collagen type II by FPC and SMC (up to 472-fold) following pellet culture. In all models, tissues generated by AC contained the highest fractions of GAG (up to 1.9% of wet weight) and were positively stained for collagen type II (specific of the inner avascular region of meniscus), type IV (mainly present in the outer vascularized region of meniscus) and types I, III and VI (common to both meniscus regions). Instead, inner meniscus, FPC and SMC developed tissues containing negligible GAG and no detectable collagen type II protein. Tissues generated by AC remained biochemically and phenotypically stable upon ectopic implantation. CONCLUSIONS: Under our experimental conditions, only AC generated tissues containing relevant amounts of GAG and with cell phenotypes compatible with those of the inner and outer meniscus regions. Instead, the other investigated cell sources formed tissues resembling only the outer region of meniscus. It remains to be determined whether grafts based on AC will have the ability to reach the complex structural and functional organization typical of meniscus tissue.

A tissue engineering approach to meniscus regeneration in a sheep model.

OBJECTIVE: Regeneration of the meniscal tissue occurs to a limited extent, and the loss of meniscal tissue leads to osteoarthritis. A new biomaterial consisting of hyaluronic acid and polycaprolactone was used as a meniscus substitute in sheep to evaluate the properties of the implant material with regard to size, biomechanical stability, tissue ingrowth, and integration. METHODS: Eight sheep (right stifle joints) were treated with three total and three partial meniscus replacements while two meniscectomies served as empty controls. The animals were euthanized after 6 weeks. The specimens were assessed by gross inspection and histology, and compared with the nonoperated left joints. RESULTS: The surgical technique was found to be feasible. The implants remained in position, did not tear, and showed excellent tissue ingrowth to the capsule. Tissue integration was also observed between the original meniscus and the implant. However, graft compression and extrusion occurred. The histological investigation revealed tissue formation, cellular infiltration and vascularization. Cartilage degeneration was more severe in the operated joints. CONCLUSION: The present study shows promising results concerning the qualities of this biomaterial with regard to implantation technique, stability and tissue ingrowth.

Analysis of mesenchymal stem cells grown on a three-dimensional HYAFF 11-based prototype ligament scaffold.

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.

Adjacent tissues (cartilage, bone) affect the functional integration of engineered calf cartilage in vitro.

OBJECTIVE: An in vitro model was used to test the hypothesis that culture time and adjacent tissue structure and composition affected chondrogenesis and integrative repair in engineered cartilage. METHOD: Engineered constructs made of bovine calf chondrocytes and hyaluronan benzyl ester non-woven mesh were press-fitted into adjacent tissue rings made of articular cartilage (AC), devitalized bone (DB), or vital bone (VB) and cultured in rotating bioreactors for up to 8 weeks. Structure (light and electron microscopy), biomechanical properties (interfacial adhesive strength, construct compressive modulus), biochemical composition (construct glycosaminoglycans (GAG), collagen, and cells), and adjacent tissue diffusivity were assessed. RESULTS: Engineered constructs were comprised predominately of hyaline cartilage, and appeared either closely apposed to adjacent cartilage or functionally interdigitated with adjacent bone due to interfacial deposition of extracellular matrix. An increase in culture time significantly improved construct adhesive strength (P<0.001), modulus (P=0.02), GAG (P=0.04) and cellularity (P<0.001). The type of adjacent tissue significantly affected construct adhesion (P<0.001), modulus (P<0.001), GAG (P<0.001) and collagen (P<0.001). For constructs cultured in rings of cartilage, negative correlations were observed between ring GAG content (log transformed) and construct adhesion (R2=0.66, P<0.005), modulus (R2=0.49, P<0.05) and GAG (R2=0.44, P<0.05). Integrative repair was better for constructs cultured adjacent to bone than cartilage, in association with its solid architectural structure and high GAG content, and best for constructs cultured adjacent to DB, in association with its high diffusivity. CONCLUSIONS: Chondrogenesis and integrative repair in engineered cartilage improved with time and depended on adjacent tissue architecture, composition, and transport properties.

Tissue engineering for cartilage repair: in vitro properties of a hyaluronan-derivative.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage and bone repair. Such scaffolds should provide a performed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells, and stimulate the phenotype of transplanted cells. Hyaff-11 is a recently developed hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell scaffolds for tissue-engineered repair. The aim of this study was to evaluate in vitro the potential of Hyaff-11 to support the growth of human chondrocytes and to maintain their original phenotype. Our data indicate that human chondrocytes seeded on Hyaff-11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration of therapeutic potential of Hyaff-11 as a delivery vehicle in tissue-engineered repair of articular cartilage defects.

Hebbian mechanisms revealed by electrical stimulation at developing rat neuromuscular junctions.

Synapse competition and elimination are widespread developmental processes, first demonstrated at neonatal neuromuscular junctions. Action potential activity was long shown to exert a powerful influence, but mechanisms and contribution relative to other factors are still not well understood. Here we show that replacement of natural motoneuronal discharge with synchronous activity suppresses elimination of polyneuronal innervation of myofibers. This requires the simultaneous chronic conduction block (tetrodotoxin) and distal electrical stimulation of motor axons during ectopic synaptogenesis in denervated adult soleus muscle. If in fact chronic stimulation is applied without central block of motor axons, the time course of synapse elimination is as fast as in control muscles undergoing natural activity. Our findings follow the prediction of Hebb's postulate and imply that asynchronous activity drives developmental synapse elimination in muscle. They further suggest that motoneurons could become transiently synchronized during development and regeneration, helping to establish the initial polyneuronal innervation.

Studies on anterograde trophic interactions based on general muscle properties.

General properties of rat skeletal muscle (extrajunctional membrane and contractile properties) are subjected to tight physiological neural regulation, as indicated by their striking alterations (up- or down-regulation) following denervation. The main contributions of the literature concerning the nature of the neural signals which mediate this regulation, are reviewed. The physiological regulation of these general properties appears to be operated by the action potential activity evoked by motoneurons in the muscle fibres. No need to postulate the participation of nerve-borne chemical substances, acetylcholine or unidentified "trophic factors", arises from the main experimental evidence. The stronger response to denervation of extrajunctional membrane properties with respect to pure paralysis is best explained by actions of factors released during wallerian degeneration of the transected nerves.

Investigations into mechanisms modulating proliferation, differentiation, and apoptosis in cultured liver, adrenal, skin, and bone cells.

The intricate modulatory roles played by manifold hormones, growth factors, cytokines, extracellular calcium concentrations, intracellular second messengers, protein kinases, and nuclear poly(ADP-ribose) polymerase in proliferative, differentiative, and apoptotic processes have been the subject of investigations that were carried out by means of in vitro either primary or secondary/tertiary cultures of differentiated epithelial (hepatocytes, keratinocytes, and adrenocytes) and connective tissue cells (osteoblasts and fibroblasts) obtained from man and/or other mammalians. In most cases, an ad hoc model system, in which cells were floated on the top of the growth medium and, hence, could enjoy nearly normal respiratory exchanges, was used. Such a system increased cell viability and the ability of parenchymal epithelial cells to respond to extremely low concentrations of growth factors, hormones, and pharmaco-toxicological agents in a way conceivably very close to their behaviour in vivo.