Tissue engineering of vascular grafts.

The challenge of tissue engineering blood vessels with the mechanical properties of native vessels, and with the anti-thrombotic properties required is immense. Recent advances, however, indicate that the goal of providing a tissue-engineered vascular graft that will remain patent in vivo for substantial periods of time, is achievable. For instance, collagen gels have been used to fabricate a tissue in vitro that is representative of a native vessel: an acellular collagen tubular structure, when implanted as a vascular graft, was able to function, and to become populated with host cells. A completely cellular approach culturing cells into tissue sheets and wrapping these around a mandel was able to form a layered tubular structure with impressive strength. Culture of cells onto a biodegradable scaffold within a dynamic bioreactor, generated a tissue-engineered vascular graft with substantial stiffness and, when lined with endothelial cells, was able to remain patent for up to 4 weeks in vivo. In our experiments, use of a non-degradable polyurethane scaffold and culture with smooth muscle cells generated a construct with mechanical properties similar to native vessels. This composite tissue engineered vascular graft with an endothelial layer formed using fluid shear stress to align the endothelial cells, was able to remain patent with an neointima for up to 4 weeks. These results show that tissue engineering of vascular grafts has true potential for application in the clinical situation.

The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis.

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.

Differentiated chondrocytes for cartilage tissue engineering.

Trauma to the articular cartilage surface of the joint represents a challenging clinical problem due to the very limited ability of this tissue to self-repair. Moreover, repair techniques such as microfracture, which introduce cells into the joint, have unpredictable clinical outcomes as they produce a fibrocartilage tissue that degenerates with time. Alternative treatments include tissue reconstruction with autograft and allograft tissue. However, these procedures are restricted by the availability of suitable donor tissue. These limitations have been the driving force behind the emerging field of articular cartilage tissue engineering. This paper will highlight and contrast the key challenges associated with the tissue engineering of this neo-tissue using differentiated adult cells. The various components of the tissue engineering process will be described including the choice of donor cell/tissue type and the selection of scaffolds that guide the formation of tissue. The ability of the tissue engineered implants to stimulate the repair of defects in vivo will also be discussed. Tissue engineering approaches may, in the future, provide an ideal alternative to the current surgical treatments for cartilage repair.

Human articular chondrocyte adhesion and proliferation on synthetic biodegradable polymer films.

The effect of polymer chemistry on adhesion, proliferation, and morphology of human articular cartilage (HAC) chondrocytes was evaluated on synthetic degradable polymer films and tissue culture polystyrene (TCPS) as a control. Two-dimensional surfaces of poly(glycolide) (PGA), poly(L-lactide) (L-PLA), poly(D,L-lactide) (D,L-PLA), 85:15 poly(D,L-lactide-co-glycolide) (D,L-PLGA), poly(epsilon-caprolactone) (PCL), 90:10 (D,L-lactide-co-caprolactone) (D,L-PLCL), 9:91 D,L-PLCL, 40:60 L-PLCL, 67:33 poly(glycolide-co-trimethylene carbonate) (PGTMC), and poly(dioxanone) (PDO) were made by spin-casting into uniform thin films. Adhesion kinetics were studied using TCPS and PCL films and revealed that the rate of chondrocyte adhesion began to level off after 6 h. Degree of HAC chondrocyte adhesion was studied on all the substrates after 8 h, and ranged from 47 to 145% of the attachment found on TCPS. The greatest number of chondrocytes attached to PGA and 67:33 PGTMC polymer films, and attachment to PCL and L-PLA films was statistically lower than that found on PGA (p < 0.05). There was no correlation between amount of chondrocyte attachment to the substrates and the substrates' water contact angle. Chondrocytes proliferated equally well on all the substrates resulting in equivalent cell numbers on all the substrates at both day 4 and day 7 of the culture. However, these total cell numbers were reached as a result of a 88- and 42-fold expansion on PDO and PLA, respectively, which was significantly higher than the 11-fold expansion found on TCPS (p < 0.05). The greater fold expansion of the cells on PDO and L-PLA films may be attributed to the availability of space for cells to grow, since their numbers at the start of culture were fewer following the 8 h attachment period. This suggests that regardless of initial seeding density on these degradable polymer substrates (i.e., if some minimum number of cells are able to attach), they will eventually populate the surfaces of all these polymers given sufficient space and time.

A method for tissue engineering of cartilage by cell seeding on bioresorbable scaffolds.

This chapter describes a method for the in vitro generation of a 3-dimensional cartilage matrix from articular chondrocytes seeded onto a bioresorbable polymeric scaffold. This particular growth system was chosen for the subject of this chapter owing to the relative simplicity of the methods required and the ease with which the necessary materials can be obtained. The tissue produced using this protocol is a cellular, metabolically active hyaline-like matrix, containing the major cartilage constituents: sulfated proteoglycan, collagen type II, and water. It serves as a useful in vitro tool for studying the influence of various mechanical and chemical factors on cartilage metabolism, as well as providing an implantable material for in vivo cartilage repair studies.

Determination of collagen-proteoglycan interactions in vitro.

The objective of this study was to characterize the physical interactions of the molecular networks formed by mixtures of collagen and proteoglycan in vitro. Pure proteoglycan aggrecan solutions, collagen (type II) suspensions and mixtures of these molecules in varying proportions and concentrations were subjected to viscometric flow measurements using a cone-on-plate viscometer. Linear viscoelastic and non-Newtonian flow properties of these solutions and suspensions were described using a second-order statistical network theory for polymeric fluids (Zhu et al., 1991, J. Biomechanics 24, 1007-1018). This theory provides a set of material coefficients which relate the macroscopic flow behavior of the fluid to an idealized molecular network structure. The results indicated distinct differences between the flow properties of pure collagen suspensions and those of pure proteoglycan solutions. The collagen network showed much greater shear stiffness and more effective energy storage capability than the proteoglycan network. The relative proportion of collagen to proteoglycan is the dominant factor in determining the flow behavior of the mixtures. Analysis of the statistical network theory indicated that the collagen in a collagen-proteoglycan mixture enhances molecular interactions by increasing the amount of entanglement interactions and/or the strength of interaction, while aggrecan acts to reduce the number and/or strength of molecular interactions. These results characterize the physical interactions between type II collagen and aggrecan and provide some insight into their potential roles in giving articular cartilage its mechanical behavior.

An investigation into the use of polymer gel dosimetry in low dose rate brachytherapy.

An investigation has been carried out into the properties of the BANG polymer gel and its use in the dosimetry of low dose rate brachytherapy. It was discovered that the response of the gel was reproducible and linear to 10 Gy. The gel was found to be tissue equivalent with a response independent of energy to within experimental accuracy (standard error of measurement +/- 5%). The slope of the calibration curve was found to increase from 0.28 +/- 0.01 s-1 Gy-1 to 0.50 +/- 0.02 s-1 Gy-1 for an increase in monomer concentration from 6 to 9%. Absorbed dose distributions for a straight applicator containing 36 137Cs sources were measured using the gel and the results compared with measurements made with thermoluminescent dosemeters (TLDs) and calculated values. Good agreement was found for the relative measurements. The root mean square residual percentage errors were 3%, 1% and 4% for the gel and the two groups of TLDs, respectively. There were some significant differences in absolute values of absorbed dose in the gel, possibly owing to the effects of oxygen. Measurements of a complex gynaecological insert were also made and compared with isodose curves from a planning system (Helax TMS), and in areas unaffected by oxygen diffusion the isodose levels from 100 to 50% agreed to within less than 0.5 mm.

Technical difficulties overcome in the use of Lowicryl 4KM EM embedding resin.

Several technical difficulties have been overcome in the use of Lowicryl 4KM resin. In order to embed and section tissue satisfactorily in the resin, it has been found necessary to thoroughly degass the resin before infiltration and polymerisation. After irradiation with UV light, the blocks are further polymerised by exposure to daylight for 2-3 weeks and then stored under partial vacuum over dessicant.

The relationship between parafunctional masticatory activity and arthroscopically diagnosed temporomandibular joint pathology.

PURPOSE: The purpose of this investigation was to assess the relationship between parafunctional masticatory activity and arthroscopically visualized changes in patients with severe, unremitting symptoms caused by intra-articular temporomandibular joint pathology. The working hypothesis was that the presence of parafunctional activity leads to increased arthroscopically diagnosed pathology. MATERIALS AND METHODS: Temporomandibular joint arthroscopy was performed on 124 joints in 83 patients (female:male, 5.4:1; mean age, 35 years; mean duration of symptoms, 49 months) with severe symptoms unresponsive to nonsurgical management. Preoperatively, the presence of parafunctional habits (bruxism, clenching) was assessed, and joints were classified as either with or without parafunctional influences. Joints were diagnosed arthroscopically and assessed for the presence or absence of osteoarthritis, synovitis, and adhesions. Analyses were performed to determine significant relationships between parafunctional activity and the presence of osteoarthritis, synovitis, and adhesions. RESULTS: Parafunctional influences were present in 82 of 124 joints (66%). Clinically diagnosed osteoarthritis was present in 59 of 124 joints (48%) and arthroscopically diagnosed osteoarthritis was seen in 82 of 124 joints (66%). Arthroscopically, synovitis was diagnosed in 123 of 124 joints (99%) and adhesions in 93 of 124 joints (75%). Statistical analyses showed a significant relationship between parafunction and clinically diagnosed osteoarthritis, and suggested a close relationship between parafunction and arthroscopically diagnosed osteoarthritis. A significant association between clinically and arthroscopically diagnosed osteoarthritis and adhesions was also demonstrated. There also was no significant relationship detected between parafunction and the presence of synovitis or adhesions seen arthroscopically. CONCLUSIONS: It was concluded that parafunctional masticatory activity and its influence on joint loading contribute to osteoarthritis of the temporomandibular joint. Such osteoarthritis is associated with adhesions of the joint. Arthroscopically diagnosed synovitis is not specifically associated with parafunction, and it appears that numerous other causative factors may contribute to its development in the TMJ. Because abnormal joint loading is a major causative factor in cartilage degradation, biochemical and biomechanical abnormalities, and intraarticular temporomandibular pathology, clinicians must identify and address parafunctional masticatory activity during nonsurgical, surgical, and postsurgical treatment regimens.

Repair of osteochondral defects with allogeneic tissue engineered cartilage implants.

The objective of this study was to evaluate the effect of allogeneic tissue engineered cartilage implants on healing of osteochondral defects. Rabbit chondrocytes were cultured in monolayer, then seeded onto biodegradable, three-dimensional polyglycolic acid meshes. Cartilage constructs were cultured hydrodynamically to yield tissue with relatively more (mature) or less (immature) hyalinelike cartilage, as compared with adult rabbit articular cartilage. Osteochondral defects in the patellar grooves of both stifle joints either were left untreated or implanted with allogeneic tissue engineered cartilage. Histologic samples from in and around the defect sites were examined 3, 6, 9, and 12, and 24 months after surgery. By 9 months after surgery, defects sites treated with cartilage implants contained significantly greater amounts of hyalinelike cartilage with high levels of proteoglycan, and had a smooth, nonfibrillated articular surface as compared to untreated defects. In contrast, the repair tissue formed in untreated defects had fibrillated articular surfaces, significant amounts of fibrocartilage, and negligible proteoglycan. These differences between treated and untreated defects persisted through 24 months after surgery. The results of this study suggest that the treatment of osteochondral lesions with allogenic tissue engineered cartilage implants may lead to superior repair tissue than that found in untreated osteochondral lesions.