A comparative study of the epiligament of the medial collateral and anterior cruciate ligaments in the human knee: Immunohistochemical analysis of CD 34, α-smooth muscle actin and vascular endothelial growth factor in relation to epiligament theory.

BACKGROUND: This study evaluated and compared the expression of VEGF, CD34, and α-SMA in the anterior cruciate ligaments and medial collateral ligaments in healthy human knees in order to enrich the epiligament theory regarding ligament healing after injury. METHODS: Samples from the mid-substance of the anterior cruciate ligament and the medial collateral ligament of 12 fresh knee joints were used. Monoclonal antibodies against CD34, α-SMA, and VEGF were used for immunohistochemical analysis. Photomicrographs were analyzed using the ImageJ software. RESULTS: The epiligament of the anterior cruciate ligament showed slightly higher expression of CD34, α-SMA, and VEGF than the epiligament of the medial collateral ligament. Overall, among the tested markers, α-SMA expression was most pronounced in anterior cruciate ligament epiligament images and CD34 dominated in medial collateral ligament epiligament images. The intensity of DAB staining for CD34, α-SMA, and VEGF was higher in vascular areas of the epiligament than in epiligament connective tissue. CONCLUSIONS: The results illustrate that CD34, α-SMA, and VEGF are expressed in the human epiligament. The differences between the epiligament of the investigated ligaments and the fact that CD34, α-SMA, and VEGF, which are known to have a definite role in ligament healing, are predominantly expressed in the main vascular part of the ligament-epiligament complex enlarge the existing epiligament theory. Future investigations regarding better ligament healing should not overlook the epiligament tissue.

A comparative immunohistochemical and quantitative study of the epiligament of the medial collateral and anterior cruciate ligament in rat knee / Estudio inmunohistoquímico y cuantitativo del epiligamento del ligamento colateral medial y cruzado anterior en rodilla de rata

SUMMARY: The aim of the present study was to evaluate the importance of the epiligament for the difference in the healing potential of the knee anterior cruciate and medial collateral ligament. To do so, we compared the structure of the anterior cruciate and the medial collateral ligament and evaluated the differences in the expression of collagen types I, III and V in a rat knee. We have also conducted a comparative quantitative analysis of the number of cells per mm2 in the two ligaments. Tissue samples were obtained from the anterior cruciate and medial collateral ligament of 10 knee joints taken from five 8-month-old Wistar rats. We used standard hematoxylin and eosin staining, in addition to immunohistochemical staining with monoclonal antibodies against collagen types I, III and V. A semi-quantitative analysis of the expression was made through ImageJ, while Student's T-test was used for the statistical analysis. Our results showed higher expression of all collagen types in the epiligament, compared to the ligament proper and difference in the expression between the medial collateral and the anterior cruciate ligament in favor of the first. We also reported a statistically significant difference in the number of cells per mm2 between the two ligaments and their epiligaments. Our findings show a higher number of cells and a stronger expression of certain collagen types in the epiligament of the medial collateral compared to the anterior cruciate ligament, which may be related to the difference in their healing potential.

RESUMEN: El objetivo del presente estudio fue evaluar la importancia del epiligamento para la diferencia en el potencial de curación del ligamento cruzado anterior y colateral medial de la rodilla. Comparamos la estructura del ligamento cruzado anterior y el ligamento colateral medial y evaluamos las diferencias en la expresión de los tipos de colágeno I, III y V en una rodilla de rata. También se realizó un análisis cuantitativo comparativo del número de células por mm2 en los dos ligamentos. Se obtuvieron muestras de tejido del ligamento cruzado anterior y colateral medial de 10 articulaciones de rodilla tomadas de cinco ratas Wistar de 8 meses de edad. Utilizamos tinción estándar con hematoxilina y eosina, además de tinción inmunohistoquímica con anticuerpos monoclonales contra colágeno tipo I, III y V. Se realizó un análisis semicuantitativo de la expresión mediante ImageJ, mientras que para el análisis estadístico se utilizó la prueba T de Student. Nuestros resultados mostraron una mayor expresión de todos los tipos de colágeno en el epiligamento, en comparación con el ligamento y una diferencia en la expresión entre el ligamento colateral medial y el ligamento cruzado anterior. También informamos una diferencia estadísticamente significativa en el número de células por mm2 entre los dos ligamentos y sus epiligamentos. Nuestros hallazgos muestran un mayor número de células y una expresión mayor de ciertos tipos de colágeno en el epiligamento colateral medial en comparación con el ligamento cruzado anterior, lo que puede estar relacionado con la diferencia en su potencial de curación.

Increased lubricin/proteoglycan 4 gene expression and decreased modulus in medial collateral ligaments following ovariohysterectomy in the adult rabbit: Evidence consistent with aging.

This study investigated whether ovariohysterectomy (OVH) surgery to induce menopause resulted in changes to modulus, failure strain and lubricin/proteoglycan 4 (PRG4) gene expression in rabbit medial collateral ligaments (MCLs), similar to aging (Thornton et al., 2015a). The MCLs from adult rabbits that underwent OVH surgery as adolescents (15-week-old) and adults (1-year-old) were compared by evaluating mechanical behaviour (adolescent OVH, n=8; adult OVH, n=7; normal, n=7), gene expression (adolescent OVH, n=9; adult OVH, n=8; normal, n=8), and collagen and glycosaminoglycan (adolescent OVH, n=9; adult OVH, n=8; normal, n=8) and water (adolescent OVH, n=9; adult OVH, n=8; normal, n=8) content. Mechanical behaviour evaluated cyclic, static and total creep strain, and ultimate tensile strength, modulus and failure strain. The RT-qPCR assessed mRNA levels for matrix regulatory genes. Adult OVH MCLs exhibited increased cyclic creep and failure strain, and decreased modulus with increased mRNA levels for lubricin/PRG4 and collagen I compared with normal MCLs. Adolescent OVH MCLs exhibited increased cyclic, static and total creep strain with decreased mRNA levels for the progesterone receptor. Lubricin/PRG4 plays a role in the lubrication of collagen fascicles which is likely related to the decreased modulus and increased failure strain observed in ligaments from adult OVH rabbits. Progesterone and its receptor are thought to play a role in the stretching of ligaments in pelvic organ prolapse and pregnancy which is likely related to the increase in creep strain observed in ligaments from adolescent OVH rabbits. Ovariohysterectomy in adult rabbits resulted in changes that were consistent with the aging MCL.

Aging affects mechanical properties and lubricin/PRG4 gene expression in normal ligaments.

Age-related changes in ligament properties may have clinical implications for injuries in the mature athlete. Previous preclinical models documented mechanical and biochemical changes in ligaments with aging. The purpose of this study was to investigate the effect of aging on ligament properties (mechanical, molecular, biochemical) by comparing medial collateral ligaments (MCLs) from 1-year-old and 3-year-old rabbits. The MCLs underwent mechanical (n=7, 1-year-old; n=7, 3-year-old), molecular (n=8, 1-year-old; n=6, 3-year-old), collagen and glycosaminoglycan (GAG) content (n=8, 1-year-old; n=6, 3-year-old) and water content (n=8, 1-year-old; n=5, 3-year-old) assessments. Mechanical assessments evaluated total creep strain, failure strain, ultimate tensile strength and modulus. Molecular assessments using RT-qPCR evaluated gene expression for collagens, proteoglycans, hormone receptors, and matrix metalloproteinases and their inhibitors. While total creep strain and ultimate tensile strength were not affected by aging, failure strain was increased and modulus was decreased comparing MCLs from 3-year-old rabbits to those from 1-year-old rabbits. The mRNA expression levels for lubricin/proteoglycan 4 (PRG4) and tissue inhibitor of metalloproteinase-3 increased with aging; whereas, the mRNA expression levels for estrogen receptor and matrix metalloproteinase-1 decreased with aging. Collagen and GAG content assays and water content assessments did not demonstrate any age-related changes. The increased failure strain and decreased modulus with aging may have implications for increased susceptibility to ligament damage/injury with aging. Lubricin/PRG4 gene expression was affected by aging and its speculated role in ligament function may be related to interfascicular lubrication, which in turn may lead to altered mechanical function with aging and increases in potential for injury.

Distinctive collagen maturation process in fibroblasts derived from rabbit anterior cruciate ligament, medial collateral ligament, and patellar tendon in vitro.

PURPOSE: Differences in the tissue-specific collagen maturation process between tendon and ligament are still unknown. Collagen cross-link formation is crucial for the collagen maturation process. The aim of this study is to examine collagen maturation processes of anterior cruciate ligament (ACL), medial collateral ligament (MCL), and patellar tendon (PT) in vitro, in order to determine the optimal cell source for tissue engineering of ligament. METHODS: Cells derived from the ACL, MCL, and PT of New Zealand white rabbits were isolated. Each cell type was cultured for up to 4 weeks after reaching confluence. Cell-matrix layers were evaluated and compared for their morphology, collagen cross-links, and gene expression levels of lysine hydroxylase 1 and 2, lysyl oxidase (LOX), tenomodulin, collagen1A1 (Col1A1), and collagen3A1 (Col3A1). RESULTS: Transmission electron microscopy photomicrographs verified that collagen fibrils were secreted from all three types of fibroblasts. A higher ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine was evident in the ligament compared to the tendon, which was consistent with lysine hydroxylase 2/lysine hydroxylase 1 gene expression. The gene expression of LOX, which regulates the total amount of enzymatic cross-linking, and the gene expression levels of Col1A1 and Col3A1 were higher in the ACL matrix than in the MCL and PT matrices. CONCLUSION: ACL, MCL, and PT cells have distinct collagen maturation processes at the cellular level. In addition, the collagen maturation of ACL cells is not necessarily inferior to that of MCL and PT cells in that all three cell types have a good ability to synthesize collagen and induce collagen maturation. This bioactivity of ACL cells in terms of ligament-specific mature collagen induction can be applied to tissue-engineered ACL reconstruction or remnant preserving procedure with ACL reconstruction.

Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile.

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1 × 10(6) or high dose 4 × 10(6) MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1ß, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.

Characterization of proteoglycan 4 and hyaluronan composition and lubrication function of ovine synovial fluid following knee surgery.

The objective of this study was to determine changes in (1) proteoglycan 4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, and (3) cartilage boundary lubricating ability of synovial fluid (SF) from surgical sham (SHAM), anterior cruciate ligament (ACL)/medial collateral ligament (MCL) transection, and lateral meniscectomy (MEN) in a post-knee surgery ovine model. Ovine SF (oSF) was collected at euthanization 20 weeks after surgery, with the contralateral joint serving as the non-operative control. PRG4 and HA concentration in oSF was measured by sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubricating ability of oSF was measured by a cartilage-cartilage friction test. PRG4 and HA concentration in SHAM, ACL/MCL, and MEN oSF were similar in comparison to the contralateral control (CTRL) oSF. The HA MW distribution in the operated oSF for all ranges were similar to the respective CTRL oSF. The kinetic coefficients of friction in operated and CTRL oSF were similar in all groups, and were significantly lower than saline. These results indicate oSF lubricant composition and function at 20 weeks post-knee surgery were similar to contralateral CTRL, and suggest earlier time points post surgery warrant further investigation.

[Effect of vanadate on proliferation and collagen production of medial collateral ligament fibroblasts].

OBJECTIVE: To investigate the effect of vanadate on proliferation and collagen type I production of rat medial collateral ligament (MCL)fibroblasts. METHODS: A total of 12 adult male SD rats were included, weighing 350-375 g. MCL was cut into small pieces (1 mm x 1 mm x 1 mm) in aseptic conditions, and then placed and cultured in culture chambers. Fibroblasts were passaged with 0.25% trypsin. The vanadate (0, 1.0, 2.5, 5.0 ng/mL) was added in the 3rd passage MCL fibroblasts, respectively, and the samples were divided into 4 groups (0, 1.0, 2.5, 5.0 ng/mL groups). MTT was used to measure the cell proliferation. The production of collagen type I was measured by RT-PCR and ELISA. Twelve samples in each group were measured. RESULTS: In fibroblast proliferation, the absorbency values of the 1.0, 2.5, 5.0 ng/mL groups were significantly different from that of the 0 ng/mL group (P < 0.05). The absorbency values of the 0, 1.0, 2.5, and 5.0 ng/mL groups were 0.213 +/- 0.016, 0.327 +/- 0.023, 0.449 +/- 0.137, and 0.561 +/- 0.028, respectively. In collagen secretion, vanadate in 1.0, 2.5, 5.0 ng/mL groups could significantly induce the production of collagen type I (P < 0.05) in a dose-dependent manner. The expressions of collagen type I of 0-5 ng/mL groups were 0.47 +/- 0.02, 0.51 +/- 0.03, 0.60 +/- 0.01, and 0.72 +/- 0.02, respectively. There was significant difference between the 1.0, 2.5, 5.0 ng/mL groups and 0 ng mL group (P < 0.05). RT-PCR displayed a dramatic increase of band density. The ratio of band density in the 0-5 ng mL groups was 1.37 +/- 0.76, 1.97 +/- 0.53, 2.41 +/- 0.94, and 2.73 +/- 0.82, respectively. The gene expression of collagen type I in the 1.0, 2.5 and 5.0 ng/mL groups was higher than that in the 0 ng/mL group, and there was significant difference (P < 0.05). There were statistical significant differences among 1.0, 2.5 and 5.0 ng/mL groups in each index mentioned above. CONCLUSION: Vanadate can effectively induce the proliferation of fibroblasts and the production of collagen type I in vitro, which may provide a new approach to the treatment of MCL injury.

Plasticity of peptidergic innervation in healing rabbit medial collateral ligament.

BACKGROUND: Denervation substantially impairs healing of the medial collateral ligament (MCL). Because normal ligaments are sparsely innervated, we hypothesized that neuropeptide-containing neurons would sprout or proliferate after ligament transection, followed by later regression with healing, in a manner analogous to blood vessels. METHODS: We transected the right MCL in 9 mature female New Zealand white rabbits and killed 3 rabbits at 2, 6 or 14 weeks. Alternate sets of 12-mm serial sections of healing MCL scars were examined by fluorescent immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and pan-neuronal marker PGP9.5. RESULTS: Normal MCLs had few peptidergic fibres located in the epiligament in a perivascular pattern. At 2 weeks, PGP9.5-, SP-and CGRP-positive fibres had increased in the epiligament adjacent to the injury. By 6 weeks, there were increases in CGRP-and PGP9.5-positive fibres in epiligament and scar, with similar but less marked increases in SP-positive fibres. At 14 weeks, there was notable regression of immunostained peptidergic nerve fibres in the scar. CONCLUSION: This experiment shows evidence for a remarkable plasticity of ligament innervation after injury, supporting the idea that neuronal factors play a fundamental role in wound healing.

Denervation alters mRNA levels of repair-associated genes in a rabbit medial collateral ligament injury model.

Previous experiments revealed that denervation impairs healing of the MCL. This suggested the hypothesis that denervation would decrease repair-associated mRNA levels in the injured MCL when compared with normally innervated injured MCL. Adult, skeletally mature female rabbits were assigned to one of four groups: unoperated control, femoral nerve transection alone (denervated controls), MCL partial tear or denervated MCL partial tear. At three days, two weeks, six weeks or sixteen weeks post-surgery, cohorts of 6 rabbits from each experimental group were killed. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbitspecific primers. In the denervated injury group, mRNA levels for the angiogenesis-associated gene MMP-13, matrix components Collagen I and III, growth factor TGF-beta and angiogenesis inhibitors TIMP-3, and TSP-1 had all increased by two-weeks post-injury, in comparison to the non-denervated injury group (p < or = 0.01). An increased level of TSP-1 mRNA was also detected in the denervated injured group at sixteen weeks post injury (p < or = 0.01). Contrary to the initial hypothesis, denervation led to increased mRNA levels for many relevant molecules during the early stages of MCL healing. Thus, inappropriate timing of over-expression of some molecules may potentially contribute to the decreased quality of the scar tissue, particularly molecules such as TSP-1. Neuronal derived factors strongly influence the in vivo metabolic activity of ligament and scar fibroblasts in the initial phases of healing.

Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells.

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.

Indirect injury stimulates scar formation-adaptation or pathology?

In several animal models of osteoarthritis induced by cruciate ligament transection, a dense, scar-like tissue mass forms rapidly on the medial side of the knee joint. This mass mimics clinical fibrosis that sometimes occurs after joint surgery. It is unknown exactly why this medial tissue mass forms and what cells are involved in its formation. This study characterizes this medial mass by histology, biochemistry, and the expression of types I and III collagen mRNA. The medial mass is compared with the medial collateral ligament (MCL) and the MCL epiligament in anterior cruciate-transected and unoperated joints, and to normal skin and skin scar. The morphology of the medial mass resembled the epiligament and skin scar more than the MCL. The concentration of DNA and RNA and the RNA-DNA ratio were elevated dramatically in the medial mass compared with all other tissues including skin scar. However, the mRNA copy number and ratio of collagen types I and III mRNAs did not differ significantly among the medial mass, MCL, epiligament, and skin in either the control or the operated joints. The response of the medial mass, MCL, and MCL epiligament to cruciate transaction involves both hyperplasia and hypertrophy, but without a dramatic shift in cell phenotype. The medial mass may be a useful mimic or model of intraarticular adhesions, hypertrophic scars, ligament sprains, and arthrofibrosis.

ACL transection influences mRNA levels for collagen type I and TNF-alpha in MCL scar.

To assess the mRNA expression of extracellular matrix genes which might correlate with or contribute to mechanically weaker medial collateral ligament (MCL) scars in the ACL-deficient rabbit knee joint compared to those in anterior cruciate ligament (ACL) intact knee joints, a bilateral MCL injury was induced in 10 skeletally mature female NZW rabbits. As part of the same surgical procedure, the ACL was transected in one of the knees while the contralateral knee had a sham procedure. The side having the combined MCL and ACL injury was randomly assigned. After six weeks, the rabbits were euthanized. Histological assessments were performed on samples of the MCL scars from each operated knee (n = 3 animals) and mRNA levels for collagen type I, III, V, decorin, biglycan, lumican, fibromodulin, TGF-beta, IL-1, TNF-alpha, MMP-1, MMP-13, and a housekeeping gene (GAPDH) were assessed using semiquantitative RT-PCR on RNA isolated from the MCL scar tissue of the remaining animals (n = 7 animals). Levels of mRNA for each gene were normalized using the corresponding GAPDH value. Results showed that the total RNA yield (per mg wet weight) in the MCL scar of the ACL-deficient knee was significantly greater than that in the MCL scar from the ACL-intact knee. Collagen type I mRNA levels were significantly lower and mRNA levels for TNF-alpha were significantly greater in the scars of ACL-deficient knees compared to scars from ACL-intact joints. There were no significant differences between ACL-deficient and ACL-intact knees with respect to MCL scar mRNA levels for the remaining genes assessed. Histologically, the "flaw" area, which has been shown to correlate with mechanical properties in previous studies, was significantly greater in MCL scars from ACL-deficient knees than in the ACL-intact MCL scars. The mean number of cells/mm2 in MCL scars from ACL-deficient knees was significantly greater than in MCL scars from ACL-intact knees. The present study suggests that MCL scar cell metabolism is differentially influenced by the combined injury environment.

Collagen expression and biomechanical response to human recombinant transforming growth factor beta (rhTGF-beta2) in the healing rabbit MCL.

We investigated biomechanical and collagen expression in a healing bilateral rabbit medial collateral ligament (MCL) model to human recombinant transforming growth factor beta (rhTGF-beta2) at three and six weeks. Each rabbit had rhTGF-beta2 in a bioabsorbable pellet administered in one side, with the contralateral side serving as control (no rhTGF-beta2). All MCL healed with rhTGF-beta2 producing a profoundly increased scar mass at three weeks which decreased in size toward control at six weeks. In-situ hybridization demonstrated collagen expression (type I and III) no different than control at three weeks, but by six weeks elevated expression of type I was seen. Biomechanical analysis at three weeks showed no effect of rhTGF-beta2 on structural properties. However, at six weeks rhTGF-beta2 significantly inhibited both the maximum load (p < 0.05) and energy absorbed (p < 0.05) with no change in stiffness. Despite increased type I collagen expression and profound increase in early scar mass, rhTGF-beta2 did not improve the structural properties. Whether the dose or mode of delivery is responsible for decline in structural properties cannot be determined in this design. We hypothesize investigations of healing ligaments to cytokines should have biologic and biomechanical properties correlated in the same study at a minimum of two time points.

Expression of heat shock protein 47(Hsp47) mRNA levels in rabbit connective tissues during the response to injury and in pregnancy.

Hsp47 (also termed "colligin") is a 47 kDa protein that is localized in the ER and cis-Golgi vesicles of fibrocytes, chondrocytes, and other collagen-secreting cells. Under stress conditions, Hsp47 expression is upregulated as part of the heat shock/stress response that mitigates cell damage from noxious stimuli such as elevated temperature, heavy metals, and oxidative stress. Under non-stress conditions, Hsp47 functions as a collagen-specific molecular chaperone that facilitates intracellular procollagen polypeptide synthesis, and triple helix assembly in connective tissues. Previously it has been shown that levels of collagen-specific gene expression are significantly altered in ligaments, menisci, and other connective tissues of the rabbit following surgically induced injuries (increased), and during pregnancy (decreased). The present study was undertaken to determine whether expression of mRNA for the Hsp47 collagen-binding stress protein was also influenced in these experimental models. Since no sequence information was available on the rabbit Hsp47 gene, a partial cDNA for rabbit Hsp47 was first isolated and cloned using reverse transcriptase PCR (RT-PCR) with degenerate oligonucleotide primers. Rabbit Hsp47 sequence-specific primers then designed enabled analysis of Hsp47 mRNA expression in rabbit connective tissues using semiquantitative RT-PCR. It was found that Hsp47 expression is affected in a complex, tissue-specific manner by injury and pregnancy. Hsp47 transcript levels were elevated in the medial collateral ligament (MCL) of the rabbit knee following surgical induction of a gap injury. Transection of the anterior cruciate ligament (ACL), which leads to chronic progressive damage to menisci of the rabbit knee joint, was accompanied by an upregulation of Hsp47 expression in the medial and lateral menisci. Hsp47 mRNA levels were depressed during pregnancy in the kidney and ACL of primigravid adolescent rabbits, but were not altered in corneal tissue during pregnancy or in the ACL of skeletally mature multiparous females. The changes in Hsp47 transcript levels within these connective tissues following injury/pregnancy often, but not always, paralleled changes in collagen-specific gene expression.

Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments: the use of different viral vectors and the effects of injury.

Gene therapy is a technique that may offer advantages over current methods of cytokine delivery to ligaments. To determine if implanted genes could be expressed in normal and injured knee ligaments, the medial collateral ligament and anterior cruciate ligament were studied in 18 rabbits. A retroviral ex vivo technique using allograft medial collateral ligament and anterior cruciate ligament fibroblasts and an adenoviral in vivo technique were compared as methods for delivering the LacZ marker gene to knee ligaments. Bilateral knee surgeries were performed, and the rabbits were equally divided into three groups. Group 1 received the retrovirus and the medial collateral ligament was ruptured, Group 2 received the adenovirus and the medial collateral ligament was ruptured, and Group 3 received the adenovirus and the medial collateral ligament was not injured. The anterior cruciate ligament was not injured in any group. The medial collateral and anterior cruciate ligaments of the right knees received 10(6) allografted, transduced ligament fibroblasts or 10(9) adenovirus particles, whereas the ligaments of the left knee received a similar volume of saline solution only. Equal numbers of rabbits were killed at 10 days, 3 weeks, and 6 weeks following the procedure. Ligament samples were stained with X-gal to detect the expression of the LacZ gene product, beta-galactosidase. LacZ gene expression was evident in ruptured and uninjured medial collateral ligaments as well as in the anterior cruciate ligament. The expression lasted between 10 days and 3 weeks in the medial collateral and anterior cruciate ligaments with use of the retrovirus and between 3 and 6 weeks in the medial collateral ligament and at least 6 weeks in the anterior cruciate ligament with the adenovirus. The length of gene expression in the ruptured and uninjured medial collateral ligaments did not differ. These preliminary studies indicate that gene transfer to normal and injured knee ligaments is possible.

Effect of local application of basic fibroblast growth factor on ligament healing in rabbits.

OBJECTIVE: The effect of basic fibroblast growth factor (bFGF) on healing of a 4-mm defect in the medial collateral ligament of rabbits was studied. METHODS: Fibrin gel containing 0 (vehicle only), 0.1, 1, or 10 micrograms of recombinant human bFGF was applied to the defect during the surgical procedure. Controls did not receive fibrin gel. Four rabbits in each group were sacrificed 1, 2, 3, and 6 weeks after surgery. Repair tissues were subjected to gross and histologic examinations, and expression of type I procollagen messenger RNA was evaluated using in situ hybridization after 2 and 3 weeks. RESULTS: bFGF promoted formation of repair tissue and was associated with early filling of the ligament defect. Tissue maturation was significantly delayed after 3 and 6 weeks in the high-dose bFGF groups. In the low dose group, in contrast, tissue maturation was similar to that in controls at all time points, by both gross and histologic examination. In situ hybridization studies showed that type I procollagen mRNA expression was reduced in all bFGF groups. CONCLUSION: Our data demonstrate that a single local application of bFGF promoted early formation of repair tissue in injured medial collateral ligaments. High doses of bFGF reduced repair tissue maturation, suggesting that in clinical uses the dose may play a significant role.