Sonya Shortkroff

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.

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.

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.

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.

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.

α-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.

Beta-particle dosimetry in radiation synovectomy.

Beta-particle dosimetry of various radionuclides used in the treatment of rheumatoid arthritis was estimated using Monte Carlo radiation transport simulation coupled with experiments using reactor-produced radionuclides and radiachromic film dosimeters inserted into joint phantoms and the knees of cadavers. Results are presented as absorbed dose factors (cGy-cm2/MBq-s) versus depth in a mathematical model of the rheumatoid joint which includes regions of bone, articular cartilage, joint capsule, and tissue (synovium) found in all synovial joints. The factors can be used to estimate absorbed dose and dose rate distributions in treated joints. In particular, guidance is provided for those interested in (a) a given radionuclides therapeutic range, (b) the amount of radioactivity to administer on a case-by-case basis, (c) the expected therapeutic dose to synovium, and (d) the radiation dose imparted to other, nontarget components in the joint, including bone and articular cartilage.

Intra-articular radiation synovectomy

Fifty-three knees in 44 patients with severe, chronic rheumatoid arthritis (RA) were treated by the instillation of an intra-articular radionuclide (dysprosium-165; 165Dy) coupled with a large, relatively inert carrier (ferric hydroxide macroaggregate). The extremely low leakage rates found in earlier animal experiments were confirmed in the human, with a mean leakage rate of 0.3% of the injected dose. This leakage corresponds to a total body dose of 0.4 rad and a liver dose of 2.5 rad, equivalent to a lumbosacral series of diagnostic radiographs. Eighty percent of the treated knees showed improvement, which was maintained as long as one year after treatment. There was a direct correlation between the preoperative radiographic stage and the response to treatment, with patients in Stages I and II more likely to have a good or excellent response at one year. There was also good correlation between clinical improvement and improvement in technetium-99m (99mTc)-pertechnetate flow scintigraphy. Chemical synovectomy by the instillation of appropriate radionuclides can be recommended as an effective means of reducing inflammation, effusion, and pain in patients with RA. The duration of favorable results cannot be predicted, but the results to date suggest that longevity should be comparable with that of surgical synovectomy. Like surgical synovectomy, radiation synovectomy is most effective in the early stages of the disease process, before there is extensive destruction of cartilage and bone.

Magnetic resonance relaxivity of dendrimer‐linked nitroxides

The relaxivity and bioreduction rates of eight dendrimer‐linked nitroxides varying in the number of nitroxides per molecule were measured and the potential use of these compounds as MR contrast agents was demonstrated. The T1 and T2 relaxivities, measured at room temperature and 1.5 T, varied linearly with the number of nitroxides per molecule for compounds with up to 16 nitroxides per molecule. Fourth‐generation polypropylenimide‐ (DAB) and third‐generation polyamidoamine‐ (PAMAM) dendrimer‐linked nitroxides were found to have greater relaxivity than gadolinium diethylenetriaminepentaacetic acid (Gd‐DTPA). The greater number of nitroxides per dendrimer increased relaxivity over that of a single nitroxide, allowing a decreased dose to achieve differential contrast with MR evaluations. Rates of nitroxide bioreduction were below detection threshold using EPR spectroscopy for generation 2 dendrimers and higher. A pilot assessment of in vivo cartilage uptake that compared intraarticular injection of three structurally different dendrimer‐linked nitroxides with Gd‐DTPA and with saline demonstrated high affinity of the DAB‐dendrimer‐linked nitroxides for normal rabbit articular cartilage. From these results, it is evident that target‐specific dendrimer‐linked nitroxides can be designed. Magn Reson Med 48:965–972, 2002.

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 μg/g) and muscle (0.35μg/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.