Factors affecting paramagnetic contrast enhancement in synovial fluid: effects of electrolytes, protein concentrations, and temperature on water proton relaxivities from Mn ions and Gd chelated contrast agents.

INTRODUCTION: Protein and electrolyte concentration of synovial fluid (SF) varies with the type of underlying arthritis. These characteristics can be utilized by magnetic resonance technology to provide a potentially significant diagnostic modality through quantitative assessments of inherent water relaxation rates and their response to contrast agents. METHODS: We evaluated the effect of a classic "in vitro" contrast agent, the Mn ion, and a common "in vivo" gadolinium based contrast agent, gadopentetate dimeglumine, on the water relaxation times of solutions with biochemical compositions simulating different types of arthritis along with similar studies of SF obtained from patients. RESULTS: The results demonstrate how protein and electrolyte concentrations play a significant role in the response of water relaxation to the Mn ion but much less so to chelated gadolinium contrast agents used clinically. DISCUSSION: A major challenge remains to develop paramagnetic agents with less toxicity than the Mn ion but with similar properties that can then serve as a tool to determine protein concentrations through imaging and thereby assist in the diagnosis of inflammatory arthrides and evaluation of therapeutic regimens.

In vivo prompt gamma neutron activation analysis for the screening of boron-10 distribution in a rabbit knee: a simulation study.

Boron neutron capture synovectomy (BNCS) is under development as a potential treatment modality for rheumatoid arthritis (RA). RA is characterized by the inflammation of the synovium (the membrane lining articular joints), which leads to pain and a restricted range of motion. BNCS is a two-part procedure involving the injection of a boronated compound directly into the diseased joint followed by irradiation with a low-energy neutron beam. The neutron capture reactions taking place in the synovium deliver a local, high-linear energy transfer (LET) dose aimed at destroying the inflamed synovial membrane. For successful treatment via BNCS, a boron-labeled compound exhibiting both high synovial uptake and long retention time is necessary. Currently, the in vivo uptake behavior of potentially useful boronated compounds is evaluated in the knee joints of rabbits in which arthritis has been induced. This strategy involves the sacrifice and dissection of a large number of animals. An in vivo (10)B screening approach is therefore under investigation with the goal of significantly reducing the number of animals needed for compound evaluation via dissection studies. The 'in vivo prompt gamma neutron activation analysis' (IVPGNAA) approach uses a narrow neutron beam to irradiate the knee from several angular positions following the intra-articular injection of a boronated compound whose uptake characteristics are unknown. A high-purity germanium detector collects the 478 keV gamma photons produced by the (10)B capture reactions. The (10)B distribution in the knee is then reconstructed by solving a system of simultaneous equations using a weighted least squares algorithm. To study the practical feasibility of IVPGNAA, simulation data were generated with the Monte Carlo N-particle transport code. The boron-containing region of a rabbit knee was partitioned into 8 compartments, and the (10)B prompt gamma signals were tallied from 16 angular positions. Results demonstrate that for this level of spatial resolution, an estimate of (10)B distribution inside the joint can be obtained to within 10% uncertainty, under ideal conditions. Variations of the anatomic dimensions among individual rabbit knees and potential knee positioning errors will result in an uncertainty of over 20%. IVPGNAA thus provides sufficient resolution and quantification regarding the in vivo uptake characteristics of boronated pharmaceuticals to serve as a useful means of screening new compounds of potential use in BNCS.

Targeted dendrimer-based contrast agents for articular cartilage assessment by MR imaging.

OBJECTIVE: Magnetic resonance (MR) imaging with contrast media has shown promise for articular cartilage assessment. Dendrimer-linked nitroxides, a new family of MR contrast agents targeted to glycosaminoglycan, may improve cartilage evaluation. This study is designed to determine the ability of dendrimer-linked nitroxides to enhance articular cartilage and measure the intra-articular life-time of these agents. DESIGN: Cartilage T(1) was evaluated using immature bovine patella in solutions of five different dendrimer-linked nitroxides, saline or Gd-DTPA at 1.5T. The "relaxivity per dose" (change in cartilage 1/T(1) produced by a given concentration of agent) was calculated. The half-life of joint fluid enhancement was measured at 2T after solutions of three dendrimer-linked nitroxides, Gd-DTPA, and saline were injected into rabbit stifle joints. Twenty-four hours after injection, the joints were examined grossly and by histology for toxicity. RESULTS: All but the largest dendrimer-linked nitroxide were able to intensely enhance articular cartilage on MR. Relaxivity per dose measurements were between 3.5 and 68 times greater than Gd-DTPA. The largest nitroxide appeared to be excluded from articular cartilage. Intra-articular half-lives of the dendrimer-linked nitroxides were sufficiently long (160-208 min) for in vivo MR imaging to be performed. Histological assessments of joints showed minimal synovial inflammatory and necrosis scores 1 day post-injection that were similar for all agents, including Gd-DTPA. CONCLUSION: Dendrimer-linked nitroxides strongly enhance cartilage and are promising as articular cartilage-specific MR contrast agents. The intra-articular life-time is sufficient for imaging studies and, in initial evaluation, the agents exhibit minimal toxicity in rabbit joints.

Alteration of matrix glycosaminoglycans diminishes articular chondrocytes' response to a canonical Wnt signal.

OBJECTIVE: Although Wnt signaling is a key regulator of the chondrocyte life cycle during embryonic development, little is known about Wnt activity in articular cartilage. Recent studies have suggested an association between excess signaling through the canonical Wnt pathway and osteoarthritis (OA). Genetic and in vitro studies with Drosophila have shown that signaling by the orthologous protein, Wingless (Wg), is regulated by glycosaminoglycans (GAGs) found at the cell surface. The objective of this study was to determine whether alteration in GAG sulfation or matrix content, such as that occurs in OA cartilage, would affect articular chondrocytes' response to a canonical Wnt stimulus. METHODS: Cells were isolated from shoulder joints of young calves (bovine articular chondrocytes, bACs) and from human cartilage (human articular chondrocytes, hACs) discarded during total knee replacement for OA. Conditioned media from a cell line that is stably transfected with Wnt3a was used as a source of Wnt protein that activates the canonical signaling pathway. Conditioned media from the parental cell line was used as a control. beta-catenin levels were measured by immunoblot. In some experiments, chondrocyte cultures were treated with sodium chlorate (NaClO3) to inhibit GAG sulfation, or with chondroitinase ABC (ChABC) to digest chondroitin sulfate (CS) in the matrix. RESULTS: Cultured bACs showed low steady-state levels of beta-catenin that increased upon stimulation with Wnt3a. A decrease in either GAG sulfation or CS content diminished bACs' response to Wnt3a (approximately 40% and 37% of control, respectively). Similar effects on the response to Wnt3a via beta-catenin were observed for cultured hACs with undersulfation of GAGs (16% of control) and decreased CS content (20% of control). CONCLUSION: This study demonstrates that articular chondrocytes respond to canonical Wnt stimulation, and that reduced sulfation or CS content diminishes that response.

Investigation of the activation of a human serum complement protein, C3, by orthopedic prosthetic particulates.

Myriad molecular, cellular, and physiological processes underlie the inflammatory and osteolytic processes induced by particles of biomaterials resulting from the wear of implants such as total joint replacement prostheses. The objective this study was to investigate the role that the complement system may be playing in these phenomena. The aim was to evaluate the degree to which particles of selected orthopaedic materials--high density and ultrahigh molecular weight polyethylene, polymethylmethacrylate, and commercially pure titanium--cause the elevation of a key complement molecule, C3a, in an in vitro assay that directly measured the concentration of C3a. The results demonstrated that HDPE particles, at high concentration, are capable of causing the elevation of C3a in the in vitro assay. This finding is discussed in the context of other work and the mechanics of the complement system as it may affect the osteolytic process.

Effect of mechanical perturbation on the release of PGE(2) by macrophages in vitro.

Macrophages play numerous roles in both physiologic and pathologic processes. Along with fibroblasts, they comprise the synovial tissue that forms the lining of musculoskeletal joint capsules and bursae, and they often envelop implants. During the process of phagocytosing prosthesis-related particles, macrophages in peri-implant tissue release inflammatory mediators. Little is known, however, about the response of these cells to mechanical perturbation, which often is a component of the physical environment of the cell. Mouse peritoneal macrophages were grown on a flexible membrane in vitro and a dynamic 1-Hz spatially uniform sinusoidal strain pattern imparted to the elastomeric substrate. The effect of mechanical strain on prostaglandin (PG) E(2) release was evaluated using cells that were activated by lipopolysaccharide (LPS) as well as by those that were not. The results are compared with the levels of PGE(2) stimulated by metallic particles. Strain magnitudes of 4 and 8% applied for 1 h resulted in almost a twofold increase in the release of PGE(2) from LPS-stimulated cells (p < 0.05) and nonstimulated macrophages (p < 0.07), compared with nonperturbated controls. No release was elicited by a challenge of metal particles. These findings demonstrate for the first time an effect of mechanical force on the release of an inflammatory mediator by macrophages. This response may help to explain the macrophage-mediated processes underlying the osteolysis associated with loose prostheses in bone and suggests a mechanism for the inflammation of synovial tissues by excessive mechanical strain.

Connective tissue orientation around dental implants in a canine model.

The objective of this study was to evaluate the orientation of collagen in the canine gingival connective tissue to a titanium surface (TI), and to hydroxyapatite coatings applied by plasma-spraying (HAPS) and ion beam assisted deposition (IBAD), on the supracrestal region of dental implants after 3 and 4 months. The effects of induced peri-implantitis on the soft connective tissue apposed to the implant were also evaluated. The use of these three surface types allowed for the evaluation of the effects of chemical composition (TI vs. IBAD; same topography) and topography (HAPS vs. IBAD; similar chemical composition) on the collagen fiber orientation. The majority of collagen fibers were found to be parallel to the implant surface, and there was no significant effect of surface type on orientation. There was a significant effect of inflammation on the connective tissue attachment length. The loss of bone height and concomitant gingival recession reduced the length of soft connective tissue apposed to the implant zone designed for collagen fiber attachment.

Autologous chondrocyte implantation in a canine model: change in composition of reparative tissue with time.

The objective of the study was to evaluate the tissue types filling 4-mm diameter defects in the canine trochlear groove 1.5, 3, and 6 months after autologous chondrocyte implantation (ACI). Untreated defects served as controls. Periosteum alone controls were also included at the 1.5-month time period. The results were compared with previously published findings obtained 12 and 18 months postoperative. After 3 months the ACI-treated defects contained significantly more reparative tissue than found in the untreated control group, including twice the amount of hyaline cartilage (HC). These findings, however, were the only significant effects of the ACI treatment when compared to the periosteum alone or empty control groups. The benefits of ACI found at 3 months did not persist to longer time periods. An evaluation of the inter-observer error associated with the histomorphometric method indicated that it was generally less than the inter-animal variation in the results.

Outgrowth of chondrocytes from human articular cartilage explants and expression of alpha-smooth muscle actin.

The objectives of this study were to investigate the effect of various enzymatic treatments on the outgrowth of chondrocytes from explants of adult human articular cartilage and the expression of a specific contractile protein isoform, alpha-smooth muscle actin, known to facilitate wound closure in other connective tissues. Explants of articular cartilage were prepared from specimens obtained from patients undergoing total joint arthroplasty. The time to cell outgrowth in vitro was determined and the expression of alpha-smooth muscle actin shown by immunohistochemistry. Treatment of the explants with collagenase for 15 minutes reduced the time to outgrowth from more than 30 days to 3 days. Hyaluronidase, chondroitinase ABC, and trypsin applied for the 15-minute period had no effect on the time to cell outgrowth when compared with untreated controls. Pretreatment with hyaluronidase prior to collagenase reduced the time to outgrowth. A notable finding of this study was that the majority of chondrocytes in the adult human articular cartilage specimens and virtually all of the outgrowing cells contained alpha-smooth muscle actin. We conclude that human articular chondrocytes have the capability to migrate through enzymatically degraded matrix and express a contractile actin isoform. Collagenase treatment reduces the time required for cell outgrowth.

Association of fibroblast orientation around titanium in vitro with expression of a muscle actin.

The objective of this study was to investigate the association of cell orientation around a biomaterial with expression of a contractile actin isoform. Selected cytokines and a fungal metabolite known to alter the cytoskeleton were used to modulate the fibroblast orientation around titanium in vitro and the synthesis of a specific muscle actin in order to reveal an association between these processes. A novel culture system using a fibronectin-coated silicone surface was employed to evaluate the orientation of human gingival fibroblasts around titanium discs. Round glass cover slips, 25 mm in diameter, were coated with polydimethylsiloxane. During the heat-induced polymerization process, two commercially pure titanium discs, 5 mm in diameter, were placed on the silicone at a distance of approximately 0.5 mm apart. The rubbery consistency of the silicone stabilized the metal discs on the cover slip and eliminated the risk of developing a lip at the edge of the titanium sample. The cover slip was then heated to complete polymerization of the silicone and subsequently coated with fibronectin. One hundred thousand human gingival fibroblasts were plated onto each glass cover slip containing the titanium discs. The cells were treated with one of the following prior to seeding on the cover slips: transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor-BB (PDGF-BB), interferon-gamma (IFN-gamma) for cytochalasin-D. Untreated cells served as controls. The orientation of the cells at the surface of the titanium discs was evaluated microscopically and the cell content of alpha-smooth muscle actin (SMA) was determined by Western blot analysis and immunohistochemistry. A notable finding was the high correlation between the percentage of cells oriented perpendicular to the titanium surface and SMA synthesis. TGF-beta1, IFN-gamma and cytochalasin-D increased synthesis of SMA while PDGF-BB decreased it. The findings support the proposition that SMA-enabled cell contraction may play a role in the orientation of cells to a biomaterial surface.

Magnetic resonance imaging-guided focused ultrasound synovectomy.

OBJECTIVE: To investigate the feasibility of magnetic resonance imaging (MRI)-guided high power focused ultrasound (FUS) to perform synovectomy noninvasively. METHODS: Five New Zealand white male rabbit knees with experimentally induced arthritis underwent MRI-guided thermal surgery by high power (60 W/10 s) sonication. Evidence of tissue coagulation was monitored during the procedure and confirmed by gross and microscopic evaluation and MRI. RESULTS: Partial synovectomy was performed in five animals. Necrotized synovial tissue was observed on gross and microscopic evaluation. Visible signal intensity alterations including high signal intensity on T2-weighted (T2W) images and lack of contrast-enhancement on T1-weighted (T1W) post-contrast, post-sonication images were characteristic and reproducible. CONCLUSION: Our results demonstrate the ability of high power sonication to destroy synovial tissue in vivo.

alpha-smooth muscle actin and contractile behavior of bovine meniscus cells seeded in type I and type II collagen-GAG matrices.

Many types of injuries to the meniscus of the knee joint result in defects that do not heal, leading to pain and dysfunction. Several ongoing investigations are developing porous absorbable matrices to be used alone or seeded with cultured cells to facilitate regeneration of this tissue. The objective of this study was to evaluate in vitro the contractile behavior of meniscal cells seeded in type I and type II collagen matrices. In many connective tissues, fibroblasts that have assumed a contractile phenotype (myofibroblasts) have been found to play an important role in healing and in pathological conditions. This phenotype, if expressed by meniscal cells, could affect their behavior in cell-seeded matrices developed for tissue engineering. In this study, the presence of a contractile actin isoform, alpha-smooth muscle (alpha-SM) actin, was assessed by immunohistochemistry in normal calf meniscal tissue and in meniscal cells in 2- and 3-dimensional culture. Calf meniscus cells were seeded in type I and type II collagen-glycosaminoglycan (GAG) matrices. The diameter of the matrices was measured every 2-3 days. Immunohistochemical staining of the 2-dimensional cultures for alpha-SM actin was performed after 1, 3, and 7 days and the staining of the seeded matrices was at 1, 7, 14, and 21 days. Transmission electron microscopy (TEM) was performed on selected samples. After 3 weeks the seeded type I matrices displayed a significant shrinkage of almost 50% whereas the type II matrix and both types of unseeded controls showed almost no contraction over the same time period. Positive staining for the alpha-SM actin phenotype was seen in 10% of the cells of the normal tissue but was present in all cells seeded in monolayer and in both types of matrices. TEM of representative cell-seeded matrices showed microfilaments approximately 7 nm thick, consistent with the myofibroblast phenotype. This is the first report of alpha-SM actin containing cells in the knee meniscus. The finding that, under certain conditions, meniscal cells can express the myofibroblast phenotype warrants study of their role in meniscal healing and the tissue response to implants to facilitate tissue regeneration.

Meniscus cells seeded in type I and type II collagen-GAG matrices in vitro.

The objective of this study was to determine the proliferative and biosynthetic activity of calf meniscus cells seeded in type I and type II collagen-glycosaminoglycan (GAG) copolymers with the overall goal to develop a cell-seeded implant for future investigations to improve the regeneration of the knee meniscus. The cell-seeded matrices were digested in protease and analyzed for GAG by a modification of the dimethyl-methylene blue method and assayed for DNA content. Other specimens were evaluated histologically after 1, 7, 14 and 21 days. Contraction of the same types of matrices, seeded with adult canine meniscus cells, was measured at the same time points. After three weeks, cells were observed throughout the type II matrix, whereas the type I matrix was densely populated at the margins. The cell morphology and the cell density after three weeks in both matrices was consistent with the normal meniscus. DNA assay for the type I matrix showed a 40% decrease over the first week and a final amount of DNA that was not significantly different from the initial value, whereas the type II matrix doubled its DNA content over the same time period. The cells continued their biosynthesis of GAG and type I collagen. GAG content of the type II matrix increased by 50% more than the type I matrix after three weeks. Over the same time period, the type I matrix displayed a significant shrinkage to approximately 50% of its initial value whereas in contrast, the type II matrix and the unseeded controls showed no significant shrinkage. The number of cells and the higher GAG synthesis in the type II matrix, and its resistance to cell-mediated contracture, commend it for future investigation of the regeneration of meniscus in vivo.

Expression of alpha-smooth muscle actin in canine intervertebral disc cells in situ and in collagen-glycosaminoglycan matrices in vitro.

The objective of this study was to investigate the presence of a contractile actin isoform, alpha-smooth muscle actin, in annulus fibrosus cells in situ and in two and three-dimensional cultures. Annulus fibrosus cells were isolated from healthy adult dogs, serial passaged, and then injected into porous collagen-glycosaminoglycan copolymers consisting of either type-I or type-II collagen. Alpha-smooth muscle actin was detected in the cells in tissue samples and in culture by immunohistochemistry. The number of cells and glycosaminoglycan content of the matrices were determined after 1, 7, and 14 days, and the diameters of the specimens were measured every 2 days. Although few annulus fibrosus cells in vivo displayed the presence of the alpha-smooth muscle actin isoform, most cells in two-dimensional culture demonstrated this phenotype. The contractile behavior of these cells was shown by the cell-mediated contraction of type-I collagen-glycosaminoglycan scaffolds after 8 days in culture. Glycosaminoglycan production was not significantly different in the seeded type-I matrices than in the unseeded matrices, whereas the seeded type-II matrices had a significant increase in glycosaminoglycan production between days 1 and 14 compared with the unseeded controls. This is the first report of both the expression of the contractile alpha-smooth muscle actin isoform in intervertebral disc cells and the ability of the cells to contract a collagen matrix. This finding could aid in better understanding the nature of cells in the annulus.

Isolation and in vitro proliferation of chondrocytes, tenocytes, and ligament cells.

In recent years it has become possible to grow large numbers of selected cell types in vitro from relatively small tissue samples. This capability has served as the foundation for using autogenous, as well as allogeneic, cells expanded in culture for engineering tissues that display little potential for spontaneous regeneration. Cartilage, tendons, and ligaments are connective tissues that have common attributes that limit their capabilities of regeneration (Table 1). These tissues are relatively avascular, sparsely populated with cells, and their cell populations have a low mitotic activity. Because the use of allogeneic grafts brings with it concerns regarding transfer of disease and immunological rejection of the foreign material, the availability of an autologous system for regeneration of these tissues is of paramount importance for restoring joint function and preventing further degeneration. Table 1 Characteristics of Adult Tissue Articular Cartilage Ligament Cruciate Collateral Cell type Chondrocyte Fibroblast/Fibrochondrocyte Fibroblast Fibroblast Cell density Low(1.4-1.7 × 10(4) cell/mm(3))(1) Low (2.7µg DNA/mg dry wt)(5) Low (2.6µg DNA/mg dry wt)(5) Low (1.5µg DNA/mg dry wt)(5) Mitotic activity Low Low Low Low Predominant collagen type II I I I Vascularity No Low Low Low Substrate for Phenotype preservation 3D Hydrogel (viz,agarose) 2D Culture plates 2D Plates 2D Plates Repair capacity Low Low High Low.

Photodynamic synovectomy using benzoporphyrin derivative in an antigen-induced arthritis model for rheumatoid arthritis.

Experimental photodynamic therapy (PDT) has recently been adapted for the treatment of inflammatory and rheumatoid arthritis. The biodistribution of benzoporphyrin derivative monoacid ring A (BPD-MA) and the effect of percutaneous light activation via intra-articular bare cleaved optical fibers was investigated using a rabbit-antigen-induced arthritis model. Qualitative evaluation of intra-articular photosensitizer clearance was performed with laser-induced fluorescence from 0 to 6 h following intravenous injection. The compound was rapidly taken up within the joint and then cleared steadily over the 6 h interval. Biodistribution was determined by fluorescence microscopy and spectrofluoroscopic extraction techniques 3 h following intravenous injection of 2 mg/kg BPD-MA. The biodistribution study demonstrated elevated levels of BPD-MA in synovium (0.35 microgram/g) and muscle (0.35 microgram/g). Fluorescence microscopy demonstrated presence of the compound within pathologic synovium but absence of the photosensitizer within meniscus, ligament, bone and articular cartilage. Tissue effects were evaluated histologically at 2 and 4 weeks posttreatment. BPD-MA-mediated PDT caused synovial necrosis in the region of light activation in 50% of treatment knees at 2 weeks and 43% at 4 weeks. No damage to nonpathologic tissues was observed. These studies indicate that selective destruction of synovium can be achieved by the light-activated photosensitizing agent BPD-MA without damage to articular cartilage or periarticular soft tissues. PDT needs to be further evaluated to optimize treatment parameters to provide for a new minimally invasive synovectomy technique.

Chondrocyte-seeded collagen matrices implanted in a chondral defect in a canine model.

The objective of our study was to evaluate reparative tissues formed in chondral defects in an adult canine model implanted with cultured autologous articular chondrocytes seeded in type I and II collagen GAG matrices. Two defects were produced in the trochlea grooves of the knees of 21 dogs, with cartilage removed down to the tidemark. This study includes the evaluation of 36 defects distributed among five treatment groups: Group A, type II collagen matrix seeded with autologous chondrocytes under a sutured type II collagen flap; Group B, type I collagen matrices seeded with chondrocytes under a sutured fascia flap; Group C, unseeded type I collagen matrix implanted under a sutured fascia flap; Group D, fascia lata flap alone; and Group E, untreated defects. All animals were killed 15 weeks after implantation. Six other defects were created at the time of death and evaluated immediately after production as 'acute defect controls'. In three additional defects, unseeded matrices were sutured to the defect and the knee closed and reopened after 30 min to determine if early displacement of the graft was occurring; these defects served as 'acute implant controls'. The areal percentages of four tissue types in the chondral zone of the original defect were determined histomorphometrically: fibrous tissue (FT); hyaline cartilage (HC); transitional tissue (TT, including fibrocartilage); and articular cartilage (AC). New tissue formed in the remodeling subchondral bone underlying certain defects was also assessed. Bonding of the repair tissue to the subchondral plate and adjacent cartilage, and degradation of the adjacent tissues were evaluated. There were no significant differences in the tissues filling the original defect area of the sites treated with chondrocyte-seeded type I and type II matrices. Most of the tissue in the area of the original defect in all of the groups was FT and TT. The areal percentage of HC plus AC was highest in group E, with little such tissue in the cell-seeded groups, and none in groups C and D. The greatest total amount of reparative tissue, however, was found in the cell-seeded type II matrix group. Moreover, examination of the reparative tissue formed in the subchondral region of defects treated with the chondrocyte-seeded collagen matrices (Groups A and B) demonstrated that the majority of the tissue was positive for type II collagen and stained with safranin O. These results indicate an influence of the exogenous chondrocytes on the process of chondrogenesis in this site. In all groups with implants (A-D), 30(50% of the FT and TT was bonded to the adjacent cartilage. Little of this tissue (6-22%) was attached to the subchondral plate, which was only about 50% intact. Remarkable suture damage was found in sections from each group in which sutures were used. Harvest sites showed no regeneration of normal articular cartilage, 18 weeks after the biopsy procedure. Future studies need to investigate other matrix characteristics, and the effects of cell density and incubation of the seeded sponges prior to implantation on the regenerative response.

Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro.

Synthetic and natural absorbable polymers have been used as vehicles for implantation of cells into cartilage defects to promote regeneration of the articular joint surface. Implants should provide a pore structure that allows cell adhesion and growth, and not provoke inflammation or toxicity when implanted in vivo. The scaffold should be absorbable and the degradation should match the rate of tissue regeneration. To facilitate cartilage repair the chemical structure and pore architecture of the matrix should allow the seeded cells to maintain the chondrocytic phenotype, characterized by synthesis of cartilage-specific proteins. We investigated the behavior of canine chondrocytes in two spongelike matrices in vitro: a collagen-glycosaminoglycan (GAG) copolymer produced from bovine hide consisting of type I collagen and a porous scaffold made of type II collagen by extraction of porcine cartilage. Canine chondrocytes were seeded on both types of matrices and cultured for 3 h, 7 days, and 14 days. The histology of chondrocyte-seeded implants showed a significantly higher percentage of cells with spherical morphology, consistent with chondrocytic morphology, in the type II sponge at each time point. Pericellular matrix stained for proteoglycans and for type II collagen after 14 days. Biochemical analysis of the cell seeded sponges for GAG and DNA content showed increases with time. At day 14 there was a significantly higher amount of DNA and GAG in the type II matrix. This is the first study that directly compares the behavior of chondrocytes in type I and type II collagen matrices. The type II matrix may be of value as a vehicle for chondrocyte implantation on the basis of the higher percentage of chondrocytes retaining spherical morphology and greater biosynthetic activity that was reflected in the greater increase of GAG content.

Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes.

This study directly compared the behaviour of chondrocytes in porous matrices comprising different collagen types and different pore diameters. There was a dramatic difference in the morphology of the cells in the type I and type II collagen matrices. The cells in the type II collagen matrix retained their chondrocytic morphology and synthesized glycosaminoglycans, while in the type I matrix the chondrocytes displayed a fibroblastic morphology with less biosynthetic activity than those in the type II. Small pore diameter affected morphology initially in the type I matrices and showed a higher increase of DNA content, but with time the cells lost the chondrocytic morphology. Our results demonstrate the marked influence of collagen type and pore characteristics on the phenotypic expression of seeded chondrocytes.

Healing of chondral and osteochondral defects in a canine model: the role of cultured chondrocytes in regeneration of articular cartilage.

In this study a canine model was developed to investigate the nature of early healing responses to both chondral and osteochondral defects and to evaluate the tissue regenerative capacity of cultured autologous chondrocytes in chondral defects. The healing response to surgically created chondral defects was minor, with little cellular infiltration. In contrast, osteochondral defects exhibited a rapid cellular response, resulting ultimately in the formation of fibrous tissue. The lack of significant cellular activity in chondral defects suggests that an evaluation of the capacity of cultured autologous chondrocytes to regenerate articular cartilage is best studied in chondral defects using the canine model. When dedifferentiated cultured articular chondrocytes were implanted into chondral defects, islands of type II collagen staining were demonstrated in the regenerative tissue within 6 weeks. The relatively early expression of cartilage specific markers by the implanted chondrocytes, coupled with the inability of untreated chondral defects to repair or regenerate, demonstrates the utility of the canine model in evaluating novel materials for cartilage repair and regeneration.