No abstract

The extracellular matrices of connective tissues contain growth factors such as transforming growth factor (TGF)-beta1. The possibility arises, therefore, that animal connective tissues that have been excised and rendered acellular in the sterilization, lyophilization, and other preparative processes for human use may still retain active growth factors that could contribute to the clinical efficacy of the product. We therefore analyzed 4M guanidine HCl extracts of a sterilized, acellular matrix, Oasis Wound Matrix, for the presence of TGF-beta1 by a sandwich enzyme-linked immunosorbent assay using the soluble type II receptor for TGF-beta to capture the growth factor, and for biological activity by testing the capacity of the extracts to inhibit 3[H]thymidine incorporation into Mv1Lu cells (mink lung epithelial cells). The enzyme-linked immunosorbent assay determined that TGF-beta1 was present at a concentration of 710.9 +/- 157.7 pg/g dry weight of tissue in Oasis and 768.1 +/- 182.1 pg/g dry weight of tissue in SIS (porcine small intestinal submucosa), the disinfected precursor of Oasis. The growth inhibition assays demonstrated that the Oasis extracts inhibited the proliferation of Mv1Lu cells in culture, consistent with the TGF-beta1 in the material having biological activity. Most of the TGF-beta1 survives the sterilization and lyophilization processes in the preparation of the Oasis Wound Matrix, and is functional in its ability to bind to its receptor and, apparently, in its capacity to inhibit growth.

The meniscus of the knee joint is a fibrocartilage mainly composed of type I collagen and smaller amounts of type II collagen. The distribution of type II collagen in the canine meniscus and its spatial relationship to type I collagen was examined by immunohistochemistry and confocal microscopy. Dorsal and coronal slices of the mid-section of medial and lateral menisci from the knee joints of skeletally mature dogs were predigested with Streptomyces hyaluronate lyase and bacterial Protease enzyme XXIV. Monoclonal antibodies against type I collagen (CP17L) and type II collagen (II-II6B3) and an anti-type II collagen polyclonal antibody (AB759) were employed. The staining for type II collagen in the extracellular matrix of hyaline articular cartilage was diffuse without any identifiable spatial organization. In striking contrast, type II collagen in the fibrocartilage of the meniscus stained as an organized network. Type II collagen was distributed throughout the meniscus with the exception of the outer zone containing the blood vessels. Coronal and dorsal staining of the meniscus showed bundles of circumferential fibrils of type I that colocalized with type II collagen in specific sites. These bundles were enwrapped in a second organizational fibrillar system of types I and II collagen that also colocalized. Bundles of circumferential fibrils appeared in cross-section in coronal sections as dots within the interstitial spaces framed by the network of types I and II collagen of the second system. Confocal overlays showed that types I and II collagens were superimposed, suggesting a close spatial proximity between the two collagens. The cells were confined to the types I and II collagen fibrils that enwrapped the bundles. A striking feature of the radial tie fibers was patches of type II collagen without colocalized type I collagen. Our study reveals a unique network of type II collagen in fibrocartilage of the meniscus that serves as a morphological distinction between fibro- and hyaline cartilage.

A comparative study of three subgroups of meniscal transplants was undertaken in the goat model: Group 1 (autograft) involved removal and immediate reimplantation of the meniscus; Group 2, fresh meniscal allografts; and Group 3, cryopreserved (30 days) meniscal allografts. Six months after surgery, tissues were evaluated for gross degenerative changes, proteoglycan concentration (as assessed by uronic acid), water content, vascularity, histology, and cell viability. The contralateral knee served as control for all comparisons. There was no statistical difference in the amount of arthritis present and all transplants demonstrated an essentially normal peripheral vascularity compared to controls. Sections revealed reduced numbers of cells in the central portions of the transplanted menisci and these viable cells demonstrated different behavior in multiplication in tissue culture compared to contralateral controls. Grossly and microscopically, the implanted menisci differed little from the controls. The measurement of proteoglycan concentration and water content of the transplanted meniscal cartilage suggest alterations that may affect the long-term mechanical properties. The autograft specimens showed the water content was very slightly increased (3% to 6%), while the proteoglycan concentration was increased (42% in terms of uronic acid). In contrast, the water content of the fresh allograft group and the cryopreserved group was increased 12% to 24%. Proteoglycan concentration in these groups was decreased up to 56% in portions of some menisci compared to controls. Fresh and cryopreserved meniscal allografts showed peripheral healing, revascularization, cellularity, and incorporation, and grossly appeared good at 6 months in the goat model. The biochemical changes in the extracellular matrix at 6 months raises questions on the long-term function of these transplanted menisci.

No abstract

The concentration, spatial distribution, and gene expression of aggrecan in meniscus, articular cartilage, and the anterior and posterior cruciate ligaments (ACL and PCL) was determined in the knee joints of five mature dogs. An anti-serum against peptide sequences specific to the G1 domain of aggrecan was employed in competitive-inhibition ELISA of guanidine HCl extracts and immunofluorescence microscopy. Gene expression was determined by Taqman real-time PCR. The concentration of aggrecan in articular cartilage (240.1 +/- 32 nMol/g dry weight) was higher than that in meniscus (medial meniscus: 33.4 +/- 4.3 nMol/g) and ligaments (ACL: 6.8 +/- 0.9 nMol/g). Aggrecan was more concentrated in the inner than the outer zone of the meniscus. Aggrecan in meniscus showed an organized, spatial network, in contrast to its diffuse distribution in articular cartilage. Thus, differences in the concentration, gene expression, and spatial distribution of aggrecan constitute another molecular distinction between hyaline cartilage and fibrocartilage of the knee.

Processes in the repair of a crevice in the knee joint meniscus were investigated in 10 dogs. Two 2-mm cylindrical plugs from each medial meniscus were removed, rendered acellular by freezing and thawing, and then reinserted into the meniscus. Dogs were euthanized at intervals of 3-52 weeks after surgery. The crevice between the plug and meniscus at 3 weeks after surgery was filled with a tissue containing alpha-smooth muscle actin-positive cells. One year after surgery, the plug had remodeled and was populated with spindle-shaped and fibrochondrocyte-like cells. The plug had an appearance intermediate between that of hyaline and fibrocartilage at this time, with a seamless integration in sites between the remodeled plug and the surrounding meniscus. alpha-smooth muscle actin-positive cells were concentrated at the interface of the remodeled plug and adjacent meniscus and at the surface of the plug. Therefore, remodeling of both the plug and meniscal tissue and the participation of alpha-smooth muscle actin-positive cells appear essential for integration of the plug into the adjacent meniscal tissue. Cells in the superficial zone of the meniscus seem to be active in the repair process. A change in both the phenotype of the cells and the quality of the matrix toward a more hyaline state appears to be an integral part of the remodeling process in the meniscus.

The capacity of non-pepsinyred type VI collagen to oind to hyaluronan wa investigated. Type V! collagen was extracted from bovine meniscal cartilage with 6 M GuHCI and purified by extraction of PEG precipitates and dissociative Sephacryl S-500 HR chromatography. Type VI collagen, detected with a monoclonal antibody, b.Jund in 0.5 M NaCI to hyaluronan-coated micro-wells, the degree of binding being higher at 37°C than 23~C and 4°C. Incubation of type Vl collagen in competitive inhibition assays with testieular hyaluronidase digests of hyaluronan in liquid phase, reduced binding of the protein to hyaturonan-coated microwells *o background levels. Thus, non-pepslnyzed ~yr ~ v! collagen binds to hyaluronan in vitro.