Near infrared spectroscopic evaluation of biochemical and crimp properties of knee joint ligaments and patellar tendon.

Knee ligaments and tendons play an important role in stabilizing and controlling the motions of the knee. Injuries to the ligaments can lead to abnormal mechanical loading of the other supporting tissues (e.g., cartilage and meniscus) and even osteoarthritis. While the condition of knee ligaments can be examined during arthroscopic repair procedures, the arthroscopic evaluation suffers from subjectivity and poor repeatability. Near infrared spectroscopy (NIRS) is capable of non-destructively quantifying the composition and structure of collagen-rich connective tissues, such as articular cartilage and meniscus. Despite the similarities, NIRS-based evaluation of ligament composition has not been previously attempted. In this study, ligaments and patellar tendon of ten bovine stifle joints were measured with NIRS, followed by chemical and histological reference analysis. The relationship between the reference properties of the tissue and NIR spectra was investigated using partial least squares regression. NIRS was found to be sensitive towards the water (R2CV = .65) and collagen (R2CV = .57) contents, while elastin, proteoglycans, and the internal crimp structure remained undetectable. As collagen largely determines the mechanical response of ligaments, we conclude that NIRS demonstrates potential for quantitative evaluation of knee ligaments.

Differences in the expression profiles of lncRNAs and mRNAs in partially injured anterior cruciate ligament and medial collateral ligament of rabbits.

Long noncoding RNAs (lncRNAs), as a novel regulatory factor, are considered to play a vital role in various biological processes and diseases. However, the overall expression profile and biological functions of lncRNAs in the partially injured anterior cruciate ligament (ACL) and medial collateral ligament (MCL) have not been clearly explored. Partially injured models of ACL and MCL were established in 3-month-old healthy male New Zealand white rabbits. Expression of lncRNAs and mRNAs in the ligament tissue was detected by high-throughput sequencing technology, and biological functions of differentially expressed RNAs were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Validation of several differentially expressed RNAs was performed using quantitative real-time PCR (qRT-PCR). Protein-protein interaction (PPI) analysis and competitive endogenous RNA (ceRNA) prediction were used to identify interactions among hub genes and the interaction among lncRNAs, miRNAs, and mRNAs. The results showed that compared with the normal group, there were 267 mRNAs and 329 lncRNAs differentially expressed in ACL and 726 mRNAs and 609 lncRNAs in MCL in the injured group. Compared with MCL, 420 mRNAs and 470 lncRNAs were differentially expressed in ACL in the normal group; 162 mRNAs and 205 lncRNAs were differentially expressed in ACL in the injured group. Several important lncRNAs and genes were identified, namely, COL7A1, LIF, FGFR2, EPHA2, CSF1, MMP2, MMP9, SOX5, LOX, MSTRG.1737.1, MSTRG.26038.25, MSTRG.20209.5, MSTRG.22764.1, and MSTRG.18113.1, which are closely related to inflammatory response, tissue damage repair, cell proliferation, differentiation, migration, and apoptosis. Further study of the functions of these genes may help to better understand the specific molecular mechanisms underlying the occurrence of endogenous repair disorders in ACL, which may provide new ideas for further exploration of effective means to promote endogenous repair of ACL injury.

Neobavaisoflavone improves medial collateral ligament-induced osteoarthritis through repressing the nuclear factor -κB/hypoxia-inducible factor-2α axis.

Osteoarthritis (OA) is a chronic inflammatory joint disease. There have been some studies on the treatment of OA with traditional Chinese medicine (TCM). Neobavaisoflavone (NBIF) is an isoflavone isolated from TCM Psoralea corylifolia L (also called 'Buguzhi') and shows anti-inflammatory effects. This study aims to explore the potential role of NBIF in treating OA. The rat chondrocytes were dealt with interleukin-1beta (IL-1ß) for inducing an in-vitro OA model and a rat OA model was established by medial collateral ligament resection. Followed by NBIF treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry were performed to evaluate chondrocyte proliferation and apoptosis. The expression of inflammatory factors and oxidative stress factors in chondrocyte medium and rat serum was tested by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining and Safranin O-Fast Green staining were carried out to examine the histopathological changes in knee joints. Caspase-3, nuclear factor-kappaB (NF-κB), and hypoxia-inducible factor-2alpha (HIF-2α) expressions were monitored by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and/or immunohistochemistry. As indicated by the results, NBIF mitigated cartilage matrix degradation and chondrocyte apoptosis in the OA rat model. NBIF hampered IL-1ß-mediated cell viability inhibition, apoptosis, inflammatory reactions, and oxidative stress of chondrocytes. Moreover, NBIF suppressed NF-κB phosphorylation and HIF-2α expression. HIF-2α overexpression induced inflammation, oxidative stress, and apoptosis in chondrocytes, while NBIF reversed HIF-2α overexpression-caused chondrocyte damage. Overall, NBIF had antiapoptotic, anti-inflammatory, and antioxidative stress effects in OA models by impeding NF-κB/HIF-2α axis, suggesting that NBIF has potential therapeutic effects in OA.

A quantitative comparison of morphological and histological characteristics of collagen in the rabbit medial collateral ligament.

Collagen fiber is one of the critical factors in determining mechanical properties of ligaments and both the morphological and histological characteristics of collagen have been widely studied. However, there was still no consensus about whether the morphological characteristics of collagen correlated with its histological characteristics in physiological ligaments. Rabbit medial collateral ligaments (MCLs) were measured under a transmission electron microscope and a polarized light microscope plus picrosirius red-staining to obtain the distributions of collagen fibril diameters and types at different anatomical sites of rabbit MCLs, respectively. The correlation between the fibril diameter and type was determined by a correlation analysis. The collagen fibril diameters at the different anatomical sites had different distributions (unimodal or bimodal) and mean fibril diameters were found to increase significantly from the anterior part to the posterior part (P=0.0482) as well as from the proximal to the distal sections (P=0.0208). Type I collagen in the core portion of MCLs was significantly less than at the other four peripheral areas (P<0.005) but no significant variation was found in each respective portion (P>0.05). The low coefficient in the correlation analysis (r=0.3759) demonstrated collagen fibril diameters had no correlation with collagen types. This may provide a new view of collagen types in studying the mechanical behavior of ligaments.

Altered cell metabolism in tissues of the knee joint in a rabbit model of Botulinum toxin A-induced quadriceps muscle weakness.

Quadriceps muscle weakness is frequently associated with knee injuries in sports. The influence of quadriceps weakness on knee joint homeostasis remains undefined. We hypothesized that quadriceps weakness will lead to tissue-specific alterations in the cell metabolism of tissues of the knee. Quadriceps weakness was induced with repetitive injections of Botulinum toxin A in six 1-year-old New Zealand White rabbits for 6 months. Five additional animals served as controls with injections of saline/dextrose. Muscle weakness was assessed by muscle wet mass, isometric knee extensor torque, and histological morphology analysis. Cell metabolism was assessed for patellar tendon, medial and lateral collateral ligament, and medial and lateral meniscus by measuring the total RNA levels and specific mRNA levels for collagen I, collagen III, MMP-1, MMP-3, MMP-13, TGF-ß, biglycan, IL-1, and bFGF by reverse transcription and polymerase chain reaction. While the total RNA levels did not change, tissue-specific mRNA levels were lower for relevant anabolic and catabolic molecules, indicating potential changes in tissue mechanical set points. Quadriceps weakness may lead to adaptations in knee joint tissue cell metabolism by altering a subset of anabolic and catabolic mRNA levels corresponding to a new functional and metabolic set point for the knee that may contribute to the high injury rate of athletes with muscle weakness.

Deficiency of macrophage migration inhibitory factor gene delays healing of the medial collateral ligament: a biomechanical and biological study.

The role played by macrophage migration inhibitory factor (MIF) in the process of wound healing is controversial. Besides, there have been no reports that investigated the expression or the role of MIF in the repair process after ligament injury. In this study, we hypothesized that the deficiency in MIF gene might delay ligament healing in mice. The aim of this study was to clarify this hypothesis using MIF gene-deficient mice (MIFKO) and murine model of injury to the medial collateral ligament (MCL). Biomechanical testing showed that the levels of mechanical properties were significantly lower in MIFKO than in wild-type mice (WT) on day 28 after injury. Levels of matrix metalloproteinase (MMP)-2 and -13 mRNA in the healing tissue were significantly lower in MIFKO than in WT on day 28 and on day 7, respectively. Histologically, healing tissues in MIFKO exhibited prolonged hypertrophy, poor vascularity, and prolonged increase in cell number compared with those in WT. Taken together, it was suggested that MIFKO exhibited delayed healing of the MCL, which might be caused by lower mRNA expression of MMP-2 and -13.

Contribution of glycosaminoglycans to the microstructural integrity of fibrillar and fiber crimps in tendons and ligaments.

The biomechanical roles of both tendons and ligaments are fulfilled by the extracellular matrix of these tissues. In particular, tension is mainly transmitted and resisted by protein (collagen, elastin) fibers, whereas compression is opposed by water-soluble glycosaminoglycans (GAGs). GAGs spanning the interfibrillar spaces and interacting with fibrils through the interfibrillar proteoglycans also seem to play a part in transmitting and resisting tensile stresses. Both tendons and ligaments showing similar composition, but different functional roles and collagen array, exhibit periodic undulations of collagen fibers or crimps. Each crimp is composed of many knots of each single fibril or fibrillar crimps. Fibrillar and fiber crimps play a mechanical role in absorbing the initial loading during elongation of both tendons and ligaments, and in recoiling fibrils and fibers when tissues have to return to their original length. This study investigated whether GAGs covalently attached to proteoglycan core proteins directly affect the 3D microstructural integrity of fibrillar crimp regions and fiber crimps in both tendons and ligaments. Achilles tendons and medial collateral ligaments of the knee from eight female Sprague-Dawley rats (90 days old) incubated in a chondroitinase ABC solution to remove GAGs were observed under a scanning electron microscope (SEM). In addition, isolated fibrils of these tissues obtained by mechanical disruption were analyzed by a transmission electron microscope (TEM). Both Achilles tendons and medial collateral ligaments of the rats after chemical or mechanical removal of GAGs still showed crimps and fibrillar crimps comparable to tissues with a normal GAG content. All fibrils in the fibrillar crimp region always twisted leftwards, thus changing their running plane, and then sharply bent, changing their course on a new plane. These data suggest that GAGs do not affect structural integrity or fibrillar crimp functions that seem mainly related to the local fibril leftward twisting and the alternating handedness of collagen from a molecular to a supramolecular level.

Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament.

Denervation degrades normal ligament properties and impairs ligament healing. This suggests that secreted neuromediators, such as neuropeptides, could be modulating cell metabolism in ligament and scar tissue. To test this hypothesis we investigated the effect of exogenous substance P (SP), neuropeptide Y (NPY) or calcitonin gene-related peptide (CGRP) on the mRNA levels for proteins associated with inflammation, angiogenesis, and matrix production in tissue-cultured specimens of normal and injured medial collateral ligament. SP and NPY induced increased mRNA levels for several inflammatory mediators in the 2-week post-injury specimens. All three neuropeptides induced decreases in mRNA levels for healing-associated growth factors and matrix molecules, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and collagen types I and III. The results indicate that neuropeptides strongly influence the metabolic activity of cells in healing ligament, particularly at early time points after injury.

Healing and normal fibroblasts exhibit differential proliferation, collagen production, alpha-SMA expression, and contraction.

This study determines the differences in proliferation, collagen production, alpha-smooth muscle actin (alpha-SMA) expression, and contraction between healing and normal fibroblasts. Transected and sham-operated rat medial collateral ligaments (MCL) were used to obtain healing and normal fibroblasts, respectively. It was found that healing fibroblasts in monolayer culture proliferated 1.4-fold faster at 48 h and had 1.7-fold greater protein expression of alpha-SMA than normal fibroblasts. In addition, it was noted that the proliferation of healing fibroblasts in collagen gels was not significantly different from that of normal fibroblasts at 24 h, but it was at 48 h. Furthermore, in collagen gels, healing fibroblasts produced more type I collagen than normal fibroblasts and generated 1.6- and 1.7-fold larger contractile forces at 15 and 20 h, respectively, than their normal counterparts. Taken together, the results of this study show that healing fibroblasts possess a differential proliferation, alpha-SMA protein expression, and contraction than normal fibroblasts.

Pathomechanics of hallux valgus: biomechanical and immunohistochemical study.

BACKGROUND: One factor believed to contribute to the development of hallux valgus is an abnormality in collagen structure and makeup of the medial collateral ligament (MCL) of the first metatarsophalangeal joint (MTPJ). We hypothesized that the mechanical properties of the MCL in feet with hallux valgus are significantly different from those in normal feet and that these differences may be related to alterations in the type or distribution of collagen fibers at the interface between the MCL and the bone. MATERIALS AND METHODS: Seven normal fresh-frozen cadaver feet were compared to four cadaver feet that had hallux valgus deformities. The MCL mechanical properties, structure of collagen fibers, and content proportion of type I and type III collagen were determined. RESULTS: The load-deformation and stress-strain curves were curvilinear with three regions: laxity, toe, and linear regions. Laxity of the MCL in feet with hallux valgus was significantly larger than that of normal feet (p = 0.022). Stiffness and tensile modulus in the toe region in feet with hallux valgus were significantly smaller than those in normal feet (p = 0.004); however, stiffness and tensile modulus in the linear region were not significantly different. The MCL collagen fibrils in the feet with hallux valgus had a more wavy distribution than the fibrils in the normal feet. CONCLUSIONS: In general, strong staining for collagen III and to a lesser extent, collagen I was observed at the interface between the MCL and bone in the feet with hallux valgus but not in the normal feet. These results indicate that the abnormal mechanical properties of the MCL in feet with hallux valgus may be related to differences in the organization of collagen I and collagen III fibrils.

Collagen fibrillogenesis and mRNA levels in the maturing rabbit medial collateral ligament and patellar tendon.

This study compared collagen fibril diameter and mRNA changes in a subset of molecules involved in collagen fibrillogenesis during postnatal development and at maturity of rabbit medial collateral ligament (MCL) and patellar tendon (PT). Tissue was analyzed by RT-PCR for mRNA levels and collagen fibril diameters were measured using transmission electron microscopy. Collagen fibril diameters increased from 3 to 14 weeks with mean fibril diameters of PT significantly greater than MCL at 9, 12, and 14 weeks and maturity. RT-PCR analysis showed decorin and lumican mRNA levels were significantly higher in PT than MCL at all ages. Type I collagen, MMP-11, and procollagen C proteinase enhancer mRNA levels also were higher in the PT than the MCL between 3 and 14 weeks but not at maturity. Further understanding of collagen fibrillogenesis by studying protein synthesis and matrix turnover during maturation may provide insight into the mechanism(s) by which fibrils accrete in maturing connective tissues and how they are altered during healing following injury.

Effects of osteogenic protein-1 (OP-1, BMP-7) on gene expression in cultured medial collateral ligament cells.

Osteogenic protein-1 (OP-1, also called BMP-7), a member of the BMP family and the TGF-beta superfamily, induces formation of new bone and cartilage, but also regulates a wide array of processes. In the present study, the expression of several characteristic biochemical markers of ligaments, such as Six1, Scleraxis, aggrecan, and type I collagen in primary cultures of adult rat medial collateral ligament (MCL) cells was determined. The effects of OP-1 on cell proliferation and on gene expression were subsequently examined. OP-1 stimulated cell proliferation, alkaline phosphatase (AP) activity, and the steady-state mRNA levels of the transcription factor Runx2/Cbfa1 in a dose- and time-dependent manner. The mRNA levels of type I collagen only increased slightly, but the activity of the cloned collagen promoter increased by 2-fold in transiently transfected MCL cells. OP-1 also stimulated aggrecan mRNA expression. The mRNA levels of Six1 and Scleraxis were not detectably altered by OP-1. In control cultures, the steady-state mRNA levels of ActR-I, BMPR-IA, BMPR-IB, and BMPR-II increased as a function of time in culture. The mRNA levels of BMP-1 and -4 increased significantly after 12 days, but those of BMP-2 and -6 did not change. The GDF-1, -3, -5, -6, and -8 mRNA levels in the control cultures also increased as a function of time. OP-1 treatment stimulated mRNA expression of BMPR-IA and BMPR-II, but had little effect on ActR-I and BMPR-IB mRNA expression. OP-1 lowered the BMP-1, -2, and -6 mRNA levels without changing the BMP-4 mRNA level. OP-1 treatment also reduced the mRNA levels of GDFs detected. In summary, the present study demonstrated that OP-1 stimulated cell proliferation and mRNA expression of several biochemical markers in this ligament cell culture model and established the spatial and temporal appearance of several members of the TGF-beta superfamily.

Investigation of the collagen fibril distribution in the medial collateral ligament in a rat knee model.

This study compared the collagen fibril diameter distribution among six anatomical sites of the rat medial collateral ligament (MCL). Ultrathin MCL sections from 4 male Sprague-Dawley rats were examined electron microscopically. With an automated quantitation method, 41,638 fibrils were measured and compared among the periphery and core regions of the femoral, middle, and tibial portions of the MCL. Results demonstrated significant difference (p < .0033) in mean fibril diameter distribution among the six sites. The mass-averaged diameters of the core and peripheral fibrils were between 175.53 to 190.82 nm and 88.47 to 109.18 nm, respectively, with the peripheral fibrils more homogeneous in size. The fibrils occupied 36.7% to 57.1% of the cross-sectional area of the ligament. About 50% of the fibrils had an oblique factor of 0.8-1.0, implying that most fibrils were aligned longitudinally. This study has provided a detailed profile of the collagen fibril distributions in rat MCL.

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.

Quantitative evaluation of nutritional pathways for the posterior cruciate ligament and the lateral collateral ligament in rabbits.

The cruciate ligament of the knee receives its nutrition from a direct vascular supply and by permeation of nutrients from the synovial fluid. The contributions of these two routes as nutritional pathways are not known in detail. In this study, we injected [3H]methyl glucose as a tracer intravenously or directly into the knee of rabbits. Tracer concentrations in plasma, synovial fluid, the posterior cruciate ligament (PCL), and the lateral collateral ligament (LCL) were analysed by a pharmacokinetic compartment model. The contribution of [3H]methyl glucose permeation from the synovial fluid during steady state was calculated at 44.3% in the PCL and at 39.0% in the LCL. Although these results indicated that more than half the nutrition for both ligaments is provided by its vascular supply, synovial fluid permeation is also an important transport route for small molecules for the PCL and the LCL, which is an extra-articular structure.

Fibroblast growth factor and epidermal growth factor receptors in ligament healing.

Although recent in vivo studies indicate that basic fibroblast growth factor hastens the healing and strength of the medical collateral ligament after injury, in vitro studies with epidermal growth factor and basic fibroblast growth factor have shown increased fibroblast proliferation with the exogenous administration of these growth factors. Using an established spontaneously healing rabbit injury model, the surgical transection of the medial collateral ligament was undertaken in 12 anesthetized male adult rabbits. Immunohistochemical localization using monoclonal antibodies to the basic fibroblast growth factor receptor and epidermal growth factor receptor were used to identify the distribution and relative concentrations of the individual receptors at 3, 7, 14, and 28 days after surgery. Realizing that the trophic effects of basic fibroblast growth factor and epidermal growth factor are determined exclusively by their individual receptors, this study confirms the hypothesis that basic fibroblast growth factor and epidermal growth factor receptor proteins are present and increase during the early stages of medial collateral ligament healing. The understanding of this rabbit ligament injury model has far reaching implications to ligament healing seen in humans. By elucidating the spatial and temporal regulation of the basic fibroblast growth factor and epidermal growth factor receptor proteins, exogenous growth factor therapy, once approved for human use, potentially can be synchronized with maximal protein receptor levels.

Morphological and immunohistochemical examination of nerves in normal and injured collateral ligaments of rat, rabbit, and human knee joints.

BACKGROUND: Knee joints possess an abundant nerve supply that relays sensory and motor information on such aspects as proprioception, nociception, and vasoregulation. Although synovial innervation has been well documented, little is known of the nerves that supply the collateral ligaments. METHODS: The morphology of rabbit and human collateral ligament nerves was examined by silver impregnation. Immunohistochemistry was performed on rabbit and rat collateral ligaments to determine the presence of peptidergic nerves in these tissues. A 6-week gap injury was performed on three rabbit medial collateral ligaments, and the localisation of peptidergic nerves in these tissues was determined. RESULTS: Irrespective of species or type of ligament examined, the greatest density of nerve fibres was found in the epiligament. Nerve fibres commonly accompanied blood vessels along the long axis of the ligament and then entered the substance of the tissue before ramifying in the deeper layers. Substance P and calcitonin gene-related peptide-immunoreactive nerve fibres were found in the collateral ligaments of the rat and rabbit. Injured ligaments showed a higher than normal level of immunoreactivity in and around the healing zone; however, the nerve fibres appeared tangled and truncated. CONCLUSIONS: Like other structures in knee joints, collateral ligaments possess a complex nerve supply. The presence of peptidergic nerves suggests that ligaments may be susceptible to neurogenic inflammation and may be centres of articular nociception.

Catabolism and loss of proteoglycans from cultures of bovine collateral ligament.

This paper investigates the kinetics and mechanism of loss of the two major proteoglycan species from cultures of bovine collateral ligament. Following incubation of ligament with [35S]sulfate after 6 days in culture, the rate of loss of the predominant proteoglycan species, decorin, from the matrix was shown to be much slower (t1/2 approximately 18 days) than that of the large chondroitin sulfate proteoglycan (t1/2 approximately 1.4 days). Analysis of 35S-labeled proteoglycans released into the medium between Days 11 and 15 of the culture period on a column of Sepharose CL-4B revealed that these macromolecules constituted mainly decorin of similar hydrodynamic size to that present in the matrix. Furthermore, analysis of core proteins using gel electrophoresis followed by fluorography or immunodetection with LF-94, an antibody directed against the amino-terminal region of decorin, indicated that the core proteins of decorin released into the medium and those remaining in the matrix of ligament cultures had a similar molecular mass (approximately 49 kDa). Analysis of both the 35S-labeled and endogenous macromolecules using 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides, revealed that three core proteins with molecular masses greater than approximately 200 kDa were present in the matrix. Four additional core proteins (range approximately 80-200 kDa) derived from the large proteoglycan were detected in the medium of ligament cultures. These findings indicate that, unlike decorin, the loss of the large chondroitin sulfate proteoglycan from the matrix of ligament cultures involved proteolytic cleavage of its core protein. No difference in the hydrodynamic size of the 35S-labeled glycosaminoglycan chains derived from either proteoglycan species remaining in the matrix or released into the medium of ligament cultures was observed.

Influence of exogenous growth factors on the synthesis and secretion of collagen types I and III by explants of normal and healing rabbit ligaments.

In this investigation it has been demonstrated that specific growth factors are able to modify collagen secretion in explants from healing rabbit medial collateral ligaments. The addition of 2.5 ng transforming growth factor beta 1 (TGF-beta 1)/mL to 3-week-old scar explants resulted in an increase in the total amount of collagen secreted. Analysis of collagen types I and III individually revealed that the increase mediated by TGF-beta 1 was due primarily to an increase in collagen type I secretion. This led to a ratio of type I:type III that is closer to that found in normal ligament tissue. The addition of 100 ng insulin-like growth factor 2 (IGF-2) to explant cultures of 3-week-old scar tissue also led to an increase in the quantity of collagen secreted, but the increase was in both type I and III collagens. These effects were observed to a lesser degree in 6-week-old scar tissue, and by 12 weeks postinjury, minimal effects of the growth factors on collagen synthesis was detected. Neither growth factor influenced collagen secretion by normal ligament or synovium. In contrast, IGF-1 (100 ng/mL) or basic fibroblast growth factor (bFGF) (10 ng/mL) did not exert a detectable effect on collagen secretion by any of the normal or healing tissues. These results indicate that TGF-beta 1 and IGF-2 can modify the metabolic activity of cells in explants of healing ligaments early after injury and may enhance the repair process leading to improved function.

Characterization of extracellular matrix macromolecules from bovine synovial capsule.

Synovial capsule from the metacarpophalangeal joints of cattle was shown to be a highly collagenous tissue, with a hydroxyproline content of 100 +/- 1 micrograms/mg dry weight and a water content of 70 +/- 3.6%. Type-I collagen made up 83% of the collagen present, and the remainder was type III. When incubated in explant culture, synovial capsule incorporated [3H]acetate into both glycoproteins and hyaluronan and [3H]acetate and [35S]sulfate into proteoglycans. The rate of synthesis of proteoglycans by synovial tissue was shown to be similar to that measured for collateral ligament from the same joint. Two populations of proteoglycans were observed to be synthesized by synovial capsule. More than 90% of the 35S-labelled proteoglycans eluted with a K(av) of 0.7 on Sepharose CL-4B, and the remainder of the radiolabelled macromolecules eluted from the column with a K(av) of less than 0.5. Analysis of the major population of proteoglycans showed it to consist of a dermatan sulfate-containing proteoglycan with a core protein of 45,000 Da that had the same N-terminal amino acid sequence as decorin.