Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4–5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61–71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.
Introduction
Some types of annelids exhibit remarkable regenerative abilities that are comparable to planarians. However, the regeneration mechanisms are thought to be quite different between planarians and annelids. Whereas planarians regenerate via totipotent stem cells (neoblasts) that are widely distributed throughout their bodies (Reddien and Alvarado 2004), annelid regeneration is thought to occur mainly through the dedifferentiation and subsequent redifferentiation of cells without any contribution from totipotent stem cells (Thouveny and Tassava 1998). In this respect, annelids regenerate in a manner similar to that of amphibians (Carlson 1998) but on a much larger scale. The elucidation of the annelid regeneration mechanisms is thus expected to provide valuable information that may allow us in the future to explore strategies to enhance the regenerative capabilities in vertebrates.
In our recent study, we have proposed the oligochaete annelid Enchytraeus japonensis as a new model system for regeneration studies (Myohara et al. 1999). E. japonensis has striking features that are highly advantageous in this regard: (1) It reproduces asexually by dividing its body into several fragments, which then regenerate a complete individual within 4–5 days, (2) artificially amputated fragments can also regenerate new individuals in the same manner, and (3) both asexual and sexual reproduction can be artificially induced in the laboratory (Myohara et al. 1999).
As an initial strategy toward the elucidation of the molecular mechanisms underlying annelid regeneration, we previously performed complementary deoxyribonucleic acid (cDNA) subtraction cloning using regenerating fragments of E. japonensis and intact animals (Myohara et al. 2006). Filter array screening subsequently revealed that about 38% of the forward-subtracted cDNA clones we isolated contained genes that were upregulated during regeneration. These clones hybridized with probes derived from regenerating fragments but not with probes from intact worms. Of these clones, 279 were then sequenced and found to contain 165 different sequences (79 known and 86 unknown). Among these 279 regeneration-upregulated clones, as many as 13 (4.7%) were found to encode glutamine synthetase, a ubiquitous and key enzyme in nitrogen metabolism (Kumada et al. 1993).
Because of its abundance in our subtracted cDNA library, we cloned the full-length sequence of E. japonensis glutamine synthetase (EjGS). By reverse transcriptase–polymerase chain reaction (RT-PCR), we subsequently revealed that the expression levels of the glutamine synthetase gene (gs) were consistently high during the regenerative process, with a gradual decline seen only in the very late stages, and our preliminary analysis by whole mount in situ hybridization further revealed strong gs expression in the blastemal tissues of the regenerating fragments (Myohara et al. 2006). As these previous results suggested an important role for gs in annelid regeneration, in the present study, we have examined the detailed expression pattern of gs in regenerating fragments and intact worms and considered the possible roles of this enzyme in the biology of E. japonensis. This is the first detailed report of the sequence and expression of gs in annelids.
Materials and methods
Animals
E. japonensis worms were reared at 24°C in 1.1% (w/v) plain agar medium in disposable Petri dishes and were fed with rolled oats as previously described (Myohara 2004). Under these conditions, the worms grow continuously to about 10 mm long, consisting of 50–60 segments, and reproduces asexually by fragmentation every 2 weeks. To induce sexual reproduction, starved worms were cultured in wet leaf mold and refed with rolled oats as previously described (Myohara 2004).
Molecular analysis
The full-length cDNA of EjGS (accession number AB109095) has been isolated in our previous study (Myohara et al. 2006). Multiple alignments of the entire amino acid sequences of the glutamine synthetase proteins from representative species, which were obtained through GenBank/EMBL/DDBJ Data Bank and chosen to cover a wide range of phylogenic groups, were performed using ClustalW software (Thompson et al 1994). To avoid errors resulting from mixing of possible pseudogenes, only data originating from full-length messenger ribonucleic acid (mRNA) sequences were used.
In situ hybridization
Both sense and antisense digoxigenin (DIG)-labeled RNA probes were synthesized using the DIG RNA labeling kit (SP6/T7, Roche) as previously described (Myohara et al. 2006). Whole-mount in situ hybridizations were also performed as previously described (Myohara et al. 2006) with some modifications to enhance the signals. Briefly, hybridization was carried out overnight with 1 μg/ml of gs DIG-labeled RNA probe at 55°C. An anti-DIG alkaline phosphatase (AP) conjugate antibody (Roche) was preabsorbed by incubation for 2.5 h with fixed and rehydrated worms and used at a dilution of 1:2,000. In situ hybridization of paraffin sections (6 μm) was carried out according to Hoshino et al. (1999) and Abe et al. (2003) with a few modifications. Briefly, sections were hybridized with DIG-labeled probes at a concentration of 100 ng/ml in probe diluent (Genostaff, Tokyo, Japan) at 60°C for 16 h. Washes were then carried out using 5× Hybri-wash (Genostaff), and an anti-DIG AP conjugate antibody (Roche Applied Science, Tokyo, Japan) was then applied at a 1:1,000 dilution in Tris-buffered saline with 0.1% Tween 20 for 2 h. After an overnight coloring reaction with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Roche), the sections were counterstained with a Kernechtrot stain solution (Muto Pure Chemicals, Tokyo, Japan), dehydrated, and mounted in Malinol (Muto Pure Chemicals).
Results and discussion
Comparisons between E. japonensis glutamine synthetase and other known glutamine synthetases
A full-length 2,717-bp cDNA encoding gs was previously isolated from regenerating E. japonensis in our laboratory (Myohara et al. 2006). The sequence of this gene was found to contain a putative open reading frame of 360 amino acids with a calculated molecular mass of 40,509 Da. Because this was the first report in annelids of a full-length sequence for gs, we performed a database search in the present study to determine whether similarities existed with the gs genes from other organisms. BLASTP analysis revealed that the deduced glutamine synthetase protein in E. japonensis (EjGS) was highly similar to known class II glutamine synthetases along its entire amino acid sequence (Fig. 1), which is well conserved across species (Kumada et al. 1993). Within the EjGS sequence, we identified a beta-grasp (catalytic) and a noncatalytic domain, five conserved regions that are common to both prokaryotes and eukaryotes, ligands for Mn2+ ions, and an adenosine triphosphate-binding site (Pesole et al. 1991). An amino acid alignment between EjGS and the gs proteins from five invertebrates, sea anemone (Cnidaria), C. elegans (Nematoda), Pacific oyster (Mollusca), fruit fly (Arthropoda), sea urchin (Echinodermata), and four vertebrates (zebrafish, clawed frog, chicken, and mouse) showed that the overall amino acid identities between EjGS and its counterparts in various other animal species were within a range of 61–71% (Fig. 1).
Fig. 1Amino acid alignment of E. japonensis glutamine synthetase (EjGS) with representative GSs from various other species. The species and protein ID of the GSs listed here are as follows: Enchytraeus japonensis (BAE93509.1), sea anemone Aiptasia pallida (AAR36878.1), nematode Caenorhabditis elegans (NP_001041010.1), Pacific oyster Crassostea gigas (CAD90162.1), fruit fly Drosophila melanogaster (AAZ41780.1), sea urchin Paracentrotus lividus (AAC41562.1), zebrafish Danio rerio (AAH66735.1), clawed frog Xenopus laevis (AAH46681.1), chicken Gallus gallus (AAA48783.1), and mouse Mus musculus (AAH15086.1). Amino acid residues are indicated in blue when greater than or equal to 70% of these residues are identical in the ten representative species. The beta grasp domain, catalytic domain, and the regions that are conserved in prokaryotes and eukaryotes are underlined by broken single lines, broken double lines, and thick single lines, respectively. Ligands for Mn2+ ions are indicated by arrowheads. The ATP-binding sites are boxed. The protein length and overall amino acid identities with EjGS are indicated at the end of each sequence
Expression of the gs gene in the blastema of regenerating E. japonensis
By whole-mount in situ hybridization analysis, we detected strong gs expression in the blastemal region in regenerating fragments of E. japonensis (Fig. 2a). This expression was detectable from the early stages of the regenerative process in the cell layer covering the wound (Fig. 2b,k) and was found to persist in the epidermal cells during the formation and elongation of the blastema (Fig. 2c,d). There was also a notable concentration of this transcript in the region corresponding to the prostomium anlagen in the anterior blastema (Fig. 2e) and in the growth zone in the posterior blastema (Fig. 2l). In situ hybridization analyses with paraffin-embedded sections additionally revealed that the gs expression pattern was mainly restricted to the epidermal cells in the early blastema (Fig. 2h) but did occur also in the inside tissues of the elongated blastema, where it was detected in the presumptive regions of the brain, ventral nerve cord, and buccal cavity (stomodeum), in addition to the prostomium and epidermal cells (Fig. 2i).
Fig. 2Glutamine synthetase mRNA expression during regeneration in E. japonensis. Transcripts were detected using whole-mount in situ hybridization (a–g and k–o) or in situ hybridization analysis of paraffin-embedded sections (h–j) using an antisense gs riboprobe, with the exception of the lower specimen shown in a. The anterior is to the left, and the ventral side is down in each image. Arrowheads indicate the amputated sites. a Whole-body images of regenerating fragments at about 1 day after amputation with the anterior blastema and an intact tail. The lower specimen was hybridized with a sense probe. b–f Lateral views of anterior blastemas at about 1 (b), 1.5 (c), 2 (d), 2.5 (e), and 4 days (f) after amputation. g Lateral view of an intact head. h–i Sagittal sections of anterior blastemas at about 1.5 (h) and 2 days (i) after amputation. j Sagittal section of an intact head. k–m Lateral views of posterior blastemas at about 0.5 (k), 1 (l), and 1.5 days (m) after amputation. n–o Ventral views of intact tails in an actively growing (n) and a full-grown individual (o). ba Brain anlage, br brain, bc buccal cavity, ch chloragocyte, ea esophageal appendage, ep epidermal cell, es esophagus, gl gut lumen, gz growth zone, m mouth, pc pharyngeal cavity, pp pharyngeal pad, pr prostomium, py pygidium, st stomodeum, va ventral nerve cord anlage, vn ventral nerve cord. Scale bars, 50 μm
In the later stages of E. japonensis regeneration, by which time segmentation and organogenesis have almost completed, we observed that the gs expression levels in the blastemal regions had become reduced (Fig. 2f,m). In the fully formed intact head, however, the expression of this gene was detectable in the prostomium, brain, anterior end of the ventral nerve cord, epithelial cells lining the buccal and pharyngeal cavities, pharyngeal pad, and esophageal appendages (Fig. 2g,j; for E. japonensis morphology, see Schmelz et al. 2000). In intact tails, gs expression was also evident in the growth zone in actively growing worms (Fig. 2n) but was absent in full-grown individuals in which the growth zone had become indistinguishable (Fig. 2o). These observations are in agreement with the RT-PCR data included in our previous report, which showed that the expression levels of gs were maintained at high levels during the early stages of regeneration, decreased temporarily in the later stages of this process, and were upregulated again in the fully formed intact worms (Myohara et al. 2006).
Expression of the gs gene in nonblastemal regions
In accordance with the abovementioned RT-PCR results, we found by in situ hybridization analysis in the present study that gs expression was not restricted to the blastemal regions (Figs. 2a and 3a) but that less intense but clear expression could be observed also in the nephridia (Fig. 3a,b), chloragocytes (Figs. 2a, 3a–c), gut epithelium (Fig. 3c), epidermis of the nonblastemal regions of regenerating fragments (Fig. 3a), and also in intact worms (Fig. 3d,e). Nephridia are the excretory organs of oligochaete annelids and the chloragocytes (chloragogen cells) are specialized peritoneal cells of the intestine that participate in the metabolism, storage, and excretion of glycogen and lipids, thus resembling the vertebrate liver in terms of function (Jamieson 1981; Edwards and Bohlen 1996). The gs expression levels in these tissues were observed to be variable among the specimens examined and also among regions within the same specimen (Figs. 2a and 3c). We were unable to find any correlation between this pattern and a specific gut region or specific condition of our specimens, although an uneven distribution of glutamine synthetase has been reported previously in the mammalian and fish digestive tracts (James et al. 1998; Mommsen et al. 2003). Intense gs expression was also found in the spermatids in the seminal vesicles and in oocytes in the ovisacs in sexually mature worms (Fig. 3e).
Fig. 3Glutamine synthetase mRNA expression in nonblastemal tissues. Transcripts were detected by in situ hybridization on paraffin-embedded sections (a–c and e) or by whole-mount in situ hybridization (d) using an antisense gs riboprobe, except for the right-hand specimen in a. The anterior is to the left, and the ventral side is down in each image. a Sagittal sections of regenerating fragments at about 0.5 days after amputation. The right-hand specimen was hybridized with a sense probe. Arrowheads indicate the amputated sites. b–c Sagittal sections of intact worms. d Whole-mount in situ hybridization analysis of an intact worm showing gs expression in the epidermal cells. e Sagittal section of a sexually mature worm. ch Chloragocyte, ep epidermal cell, ge gut epithelium, gl gut lumen, ne nephridium, oo oocyte, sc spermatocyte, st spermatid, sp sperm. Scale bars, 50 μm
General comparisons between the distribution of glutamine synthetase in different organisms
Glutamine synthetase is found in a wide range of organisms, including microorganisms, plants, and animals and is known to be expressed in a tissue-specific and developmentally controlled manner. In murine organs, glutamine synthetase activity is highest in the liver, followed in descending order by the epididymis, testes, uterine tube, stomach, kidney, brain, and adipose tissues (van Straaten et al. 2006). High glutamine synthetase expression in the brain, kidney, and liver has also been reported in fish (Murray et al. 2003). In Xenopus, gs transcripts have been detected in the ovary, kidney, eye, muscle, and embryo but not in the brain, heart, liver, stomach, or testis (Hatada et al. 1995). In the case of invertebrates, little information is currently available regarding the expression profile of this enzyme and is restricted to arthropods; gs expression has been reported in mosquito midgut epithelium, ovary, and fat body (Smartt et al. 2001) and in crustacean neural tissues (Linser et al. 1997; Allodi et al. 2006; Sullivan et al. 2007). Our present study, therefore, provides novel information regarding the expression of this enzyme in nonarthropod invertebrates, and our results are in general agreement with those of previous reports; that is, in full-grown E. japonensis, gs is expressed in (1) chloragogen tissues, which correspond to the vertebrate liver in terms of functionality (Jamieson 1981), (2) nephridia, which are the annelid counterparts of the kidney and uterine tube in vertebrates (Jamieson 1981), (3) the brain and ventral nerve cord, (4) the prostomium where large numbers of sensory cells including photoreceptor cells are present (Jamieson 1981; Edwards and Bohlen 1996), (5) the epithelium of the digestive tract, and (6) spermatids and oocytes.
Possible functions of glutamine synthetase in E. japonensis
Regeneration The possible involvement of glutamine synthetase in the regeneration of damaged tissue has been reported in only a few cases. These include the observed increase in gs expression after peripheral motor nerve injury in mouse (Toki et al. 1998) and also in Xenopus tail regeneration in which gs expression was found to be upregulated and could be detected in the terminal vesicle of the regenerating spiral cord (Tazaki et al. 2005). Our present results, together with our previously reported findings, strongly suggest the involvement of glutamine synthetase also in annelid regeneration. In the previous study, we have shown that glutamine synthetase is one of the most abundantly expressed upregulated genes during regeneration in E. japonensis (Myohara et al. 2006), and in the present study, our detailed expression analysis by in situ hybridization has revealed strong gs expression in the blastemal regions of regenerating E. japonensis. It has been reported previously in Xenopus that embryos abundantly express glutamine synthetase in the developing nervous system during the gastrula and neurula stages (Hatada et al. 1995). In accordance with this finding, we observed in the present analyses that gs is expressed in the presumptive regions of the brain and ventral nerve cord anlagen in the blastema during E. japonensis regeneration. This further suggests the involvement of glutamine synthetase in annelid neural regeneration.
Nerve function Glutamine synthetase has been shown to play an important role in glutamate transmitter degradation in the nervous system (Derouiche and Frotscher 1991) and is used as a specific glial cell marker in the neural retina (Vardimon et al. 1993). In intact E. japonensis, the expression of this enzyme was evident in the brain and ventral nerve cord, suggesting that it has a nervous system function. Its expression in the prostomium may also be attributable to the distribution of numerous sensory cells, probably including photoreceptor cells, in this region (Jamieson 1981; Edwards and Bohlen 1996).
Cell proliferation Glutamine synthetase is the only enzyme that can synthesize glutamine, which provides nitrogen for the synthesis of amino acids, purines, pyrimidines, and other compounds required in many critical cellular events including proliferation (Meister 1980). In the rat intestinal epithelium, high concentrations of glutamine synthetase protein and mRNA have been found in the highly proliferative crypt region (Roig et al. 1995). Hence, the high gs expression levels that we observed in blastemal cells and in the growth zone in actively growing worms could be explained, at least in part, by a high demand for glutamine during the intense cell proliferation that would be ongoing in these areas.
Detoxification Glutamine synthetase is also important for the detoxification of excess glutamate and ammonia by converting them to glutamine, which is nontoxic and can be stored in tissues or circulate throughout the body without causing any harm (Meister 1980). A comparison between the cellular concentrations of glutamine synthetase in various murine organs in a previous report has also revealed that the highest concentrations of this enzyme positively correlate with the cells that have detoxifying functions (van Straaten et al. 2006). The gs expression that we observed in the nephridia and chloragocytes in E. japonensis herein could therefore be ascribed to the involvement of these tissues in excretion of nitrogenous waste in oligochaete annelids (Jamieson 1981).
Macromolecule synthesis Glutamine is known to be required for the synthesis of certain macromolecules that are present in mucus, such as the hexosamines that lubricate and protect the gut mucus (Weiss et al. 1999). The mucus secreted by epidermal cells covering the earthworm body is also essential for locomotion, gas exchange, and protection in these animals (Jamieson 1981), and hexosamines have been found to be a component of their cuticles (Muir and Lee 1970). We speculate therefore that the gs transcripts in the E. japonensis epidermal cells have a role in mucus and/or cuticle synthesis in intact and regenerating individuals. In addition, the gs expression found in epithelial cells of the buccal and pharyngeal cavities may be attributed to cuticle synthesis in these tissues (Jamieson 1981).
Gametogenesis Glutamine synthetase expression in the ovary has been reported in Xenopus (Hatada et al. 1995) and in the yellow fever mosquito (Smartt et al. 2001). In oligochaete annelids, the testes and ovaries are small organs located in the ventral part of the genital segments (Edwards and Bohlen 1996). The spermatogonia that are formed in the annelid testes pass into the seminal vesicles, which contain male cells at all stages of development. Oogonia in annelids are formed in the ovaries and divide to form oocytes, which are shed from the ovaries into the ovisacs. In E. japonensis, gs transcripts were detectable in spermatids in the seminal vesicles and in oocytes in the ovisacs but not in the testes or ovaries, suggesting that glutamine synthetase may have a role in the later stage of gametogenesis.
In summary, in the present study, we have characterized an annelid gs gene and shown that it is expressed in a tissue-specific manner in regenerating and intact E. japonensis. To further elucidate the actual functions of this gene, additional studies including RNAi gene knockdown experiments will be needed in the future. Ongoing investigations in our laboratory of other regeneration-associated upregulated genes, several of which are novel (Myohara et al. 2006), should also shed important new light on the network of biological pathways involved in the regenerative processes in E. japonensis.

glutamine synthetase
gene expression
regeneration
enchytraeus japonensis
oligochaete annelid


Antioxidant intervention in rheumatoid arthritis: results of an open pilot study

There is evidence that reactive oxygen species play a causal role in auto-immune diseases, such as rheumatoid arthritis (RA). Despite the supporting evidence for a beneficial effect of antioxidants on clinical characteristics of RA, the right balance for optimal effectiveness of antioxidants is largely unknown. To determine the potential beneficial effects of an antioxidant intervention on clinical parameters for RA, an open pilot study was designed. Eight non-smoking female patients with rheumatoid factor + RA and a Disease Activity Score (DAS 28) higher than 2.5 were enrolled in the study. Patients had to be receiving stable non-steroidal anti-inflammatory drug treatment and/or ‘second line’ medication for at least 3 months. The pilot group consumed 20 g of antioxidant-enriched spread daily during a period of 10 weeks. The intervention was stopped after 10 weeks and was followed by a ‘wash-out’ period of 4 weeks. At t = 0, t = 10 weeks and t = 14 weeks, patients’ condition was assessed by means of DAS. In addition, standard laboratory analyses were performed, and blood-samples for antioxidants were taken. The antioxidant-enriched spread was well tolerated. All laboratory measures of inflammatory activity and oxidative modification were generally unchanged. However, the number of swollen and painful joints were significantly decreased and general health significantly increased, as reflected by a significantly improved (1.6) DAS at t = 10 weeks. The antioxidant effect was considered beneficial as, compared to the scores at t = 0, the DAS significantly reduced at t = 10 weeks. Increase of the DAS (0.7) after the “wash-out period” at t = 14 confirmed a causal relation between changes in clinical condition and antioxidants. This open pilot study aimed to assess the clinical relevance of an antioxidant intervention as a first step in assessing potential beneficial effects of antioxidants on rheumatoid arthritis. These conclusions need to be validated in a larger controlled study population.
Introduction
In spite of the great advances that have been made in the development of new drugs for the treatment of patients with rheumatoid arthritis (RA), many patients are interested in alternative treatments like dietary therapy. Although the aetiology of rheumatoid arthritis is still unknown, the inflammation resulting from the immunological reaction is quite well described. It is known that neutrophil granulocytes, macrophages and lymphocytes are activated, and that reactive oxygen and nitrogen species (RS) are produced [1, 2]. These RS can react with lipid, protein and nucleic acids and are thought to be of importance for the aetiology and chronicity of the inflammatory rheumatic diseases [3, 4]. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. There is some evidence for a positive effect of antioxidants on clinical symptoms of RA [5, 6]. However, this evidence is weak, and information about the most effective antioxidants, antioxidant doses or combinations is lacking The intervention studies that have been conducted have tested vitamin E exclusively [6, 7].
Against this background, an open (uncontrolled) pilot study was designed to assess if effects of antioxidants on the clinical parameters of patients with RA could be confirmed and if these effects would associate with changes in a selected number of molecular markers related to human antioxidant defence system status. Further, a mix of antioxidants was to be tested rather than a single high-dosed compound.
Materials and methods
The open study was approved by the ethical committee. Eight patients with rheumatoid factor + RA, as defined by the ARA revised criteria were enrolled in the study. To avoid influences of gender, only female patients participated. Inflammatory disease activity was defined as a Disease Activity Score (DAS) higher than 2.5. Inclusion criteria included non-smoking and no serious co-morbidity. Patients had to be receiving stable non-steroidal anti-inflammatory drug (NSAID) treatment and/or ‘second line’ medication for at least 3 months. Those taking antioxidant supplements were excluded from the study. During a period of 10 weeks, patients consumed 20 g of antioxidant-enriched margarine daily. The spread contained a mix of a-tocopherol (400 mg), lycopene (10 mg), palm oil carotenoids (5 mg; mainly α-carotene) and lutein (10 mg). Further, patients received vitamin C (200 mg daily) as a supplement.
The intervention of 10 weeks was followed by a ‘wash-out’ period of 4 weeks. At t = 0, t = 5 weeks, t = 10 weeks and t = 14 weeks, patients’ condition was assessed by means of morning stiffness, visual analogue scale pain, general health score and DAS 28-score (including the following parameters: pain, swelling of joints, erythrocyte sedimentation rate (ESR), general feeling of well-being). At t = 0, t = 10 weeks and at t = 14 weeks, blood was collected, and standard clinical laboratory analyses (ESR, C-reactive protein, hemoglobin, leucocytes, thrombocytes, serum values of creatinine and transaminases) were performed. In addition, blood antioxidant status and markers of antioxidant capacity [ferric reducing ability of plasma (FRAP)] and oxidative stress (8-epi F2-isoprostane) levels were determined. Determination of vitamin A, not present in the spread, was used as a control. Serum was collected immediately after blood sampling and stored in liquid nitrogen. The potential antioxidant effect was defined as beneficial if the DAS score would significantly improve >1.2 at t = 10 weeks as compared to the scores at t = 0. At t = 14 weeks, a ‘wash-out” effect was assessed using the same parameters. Return of the clinical condition of the pilot group to that at t = 0 may indicate a causal relation between changes in clinical condition and antioxidant intake, although a placebo-controlled intervention would be better required for this assessment.
Statistical analyses were done using a paired t test.
Results
The antioxidant-enriched spread was well tolerated. None of the patients withdrew from the study because of adverse reactions. The number of swollen and painful joints were significantly decreased, and general health significantly increased at t = 10 weeks. This was shown by a significantly decreased DAS (−1.6) at 10 weeks. After the “wash-out period”, the DAS score was increased again by 0.6 (Figs. 1 and 2).
Fig. 1Clinical condition by Disease Activity ScoreFig. 2Mean Disease Activity Score
The standard laboratory measures of inflammatory activity were relatively unchanged. Levels of plasma vitamin E, lutein, lycopene and alpha-carotene were significantly increased at 10 weeks. No significant changes were seen in vitamin A and vitamin C levels (Fig. 3).
Fig. 3Plasma antioxidant status in eight patients
Markers of antioxidant capacity and oxidative stress displayed trends for differences, but none of these reached statistical significance (Fig. 4).
Fig. 4Markers of antioxidant capacity and oxidative stress
Discussion
This pilot study was conducted to obtain some first indications for potential beneficial effects of an antioxidant intervention on clinically relevant parameters for rheumatoid arthritis. In our present study, a significant reduction in Disease Activity Score (DAS) was observed following antioxidant intervention of 10 weeks. This was accompanied by significant increases in blood levels of antioxidants administered. The reduction of DAS is remarkable as all patients had an active (high initial mean DAS of 5.8) longstanding disease and had tried all kinds of disease-modifying anti-rheumatic drugs, including combination therapy. The unchanged level of vitamin A, not present in the spread, reinforces the significance of the increased levels of the blood antioxidant status. As NSAIDs can influence the absorption of vitamin C, this could explain the non-significant changes of the serum concentration of this antioxidant.
A limited number of clinical intervention studies is available which support the therapeutic or prophylactic activity of antioxidants in the pathogenesis of RA. These studies were all conducted with an oral intake of 1,200 mg/day of vitamin E. Edmonds et al. [6] reported that the antioxidant a-tocopherol significantly reduced pain parameters in a placebo-controlled double-blind trial following a 3-week supplementation period. Results from a randomised double-blind parallel group comparison study with a-tocopherol and diclophenac showed that the clinical parameters assessed, e.g. morning stiffness, Ritchie joint index, grip strength and pain, were significantly reduced by vitamin E, with similar effectivity and less side effects as compared to regular drug therapy by diclofenac [7]. Results from other intervention studies have generally been in line with these observations [8, 9].
In our study, not only the effect of vitamin E on the inflammatory response and clinical symptoms was evaluated but also the interactions with bioavailable natural antioxidants such as carotenoids, vitamin C and others. In view of the limited number of controlled studies, the supporting evidence for beneficial effects of antioxidants on clinical characteristics of rheumatoid arthritis may be considered limited but promising. Another point is that the potential mechanism of action of antioxidants in rheumatoid arthritis needs further attention. The aspect of damage by RS and loss of critical functions can be analysed by use of ‘molecular markers’. These markers may include antioxidant status, products which arise as a consequence of oxidative damage to lipid, protein and DNA and tissue damage (as assessed by lactic dehydrogenase release) or cytokine levels (cq anti-TNFα).
Although markers are very informative from a mechanistic point of view, a drawback is that none of them has been validated against clinical conditions of patients with RA. In this study, a trend for reduced levels of measures of antioxidant capacity and of ‘footprints’ of oxidative stress was observed, e.g. statistically non-significant increase in FRAP and reduction in F2-isoprostane levels. Furthermore, these markers showed a trend for correlation with clinical conditions. The data indicate that statistically significant effects may be observed when the number of patients enrolled is increased.
This open pilot study therefore should be viewed as a first step in assessing potential beneficial effects of the antioxidants tested on rheumatoid arthritis. The data indicate there may be potential in using mixes of antioxidants in the treatment of RA alongside drug treatment so that drug dosage may be reduced while hopefully retaining efficacy. Further, multi-drug/antioxidant therapy could be used to reduce drug dose and thus side effects of treatment.
In conclusion, our present pilot study indicated that intervention with antioxidant-enriched margarine in RA results in consistent and significant relief of clinical symptoms. Furthermore, increases in blood antioxidant status and indications for effects on oxidative stress markers were observed. These data are promising and indicate need for a double-blind, placebo-controlled randomised human trial to establish effect and demonstrate causality.

antioxidants
intervention
rheumatoid arthritis
clinical parameters


Anterior cruciate ligament reconstruction: a look at prosthetics - past, present and possible future

Biological tissue autograft reconstruction using the patellar tendon or quadrupled semitendinosus/gracilis tendons has become the most popular procedure in surgical treatment of a ruptured ACL. This article provides a review of the history of the use of prosthetics with respect to ACL reconstruction grafts including Carbon Fibre, Gore-Tex and Dacron prosthetics as well as the Leeds-Keio Artificial Ligament and the Kennedy Ligament Augmentation Device (LAD). Emphasis is placed on the Ligament Advanced Reinforcement System (LARS) as preliminary investigations of its use have been encouraging. Significant progress has been made recently with respect to the understanding of ACL anatomy, composition, biomechanics, and healing processes, leading to innovative techniques using approaches based in tissue engineering principles and computer – assisted surgery. While research into improved ACL treatment options continues, the synthesis of recent advancements provides a new optimism towards the regeneration of an ACL mirroring its original stability, function, and longevity.
INTRODUCTION
The anterior cruciate ligament (ACL) is the most frequently injured ligament in the knee and consequently, the majority of research into knee ligament injuries has been directed towards the ACL. While the collateral knee ligaments exhibit strong healing potential and generally respond well to conservative treatment, the anterior cruciate ligament has a poor intrinsic healing ability due to the fact that it is enveloped by synovial fluid and lacks significant vascularization (1). Surgical reconstruction is therefore the most frequent mode of treatment pursued when the ACL is torn. The patients who experience ACL injuries are significantly younger and more active than those who experience many other orthopaedic injuries. The need for reconstruction options that exhibit longevity in the face of great stresses is therefore imperative (2). Historically, options for surgical treatment have included primary repair with or without synthetic augmentation and reconstruction using either biological tissue grafts or prosthetic ligaments. Primary repairs with or without augmentation have tended to fail at restoring stability to the knee and are not a common treatment option today (3). Likewise, prosthetic replacements have traditionally tended to be inadequate due to post-surgical complications arising from wear and degeneration. Hence, biological tissue autograft reconstruction using the patellar tendon or quadrupled semitendinosus/gracilis tendons has become the most popular procedure in surgical treatment of an ACL rupture. However, the frequency of significant anterior knee pain post-surgically and high occurrence of flexion contracture and crepitation in tendon autografts have kept research interests focused on the further development of prosthetic ligament implants. A significant number of early research endeavours into prosthetic ACL replacements failed due to a poor understanding of the biomechanical and physiological properties of the ACL. Tremendous gains in understanding have been made in these areas, with significant progress being made in understanding the inter-dependence between ACL anatomy, tissue composition, biomechanics, and the healing processes. Unfortunately, to date, no prosthesis has proven itself as a viable alternative to the patellar or hamstring tendon autografts, currently used in over 90% of ACL reconstructions. This article reviews some of the major historical milestones in ACL reconstruction technology and looks forward to the continuing evolution of this technology. The literature used for this review was obtained using the PubMED database with keywords “anterior cruciate ligament” searched in tandem separately with “prosthetics” and “tissue engineering.” It should be noted that many of the studies cited in this review obtained funding from orthopaedic and biomaterial companies that sponsored the implant.
SURGICAL REPAIR: ADVANTAGES AND DISADVANTAGES OF BIOLOGICAL TISSUE GRAFTS
Bone-patellar tendon-bone (BPTB) and semitendinosus/gracilis tendon autografts are currently the most common grafts used in ACL reconstruction. While the BPTB autografts were the former “gold standard”, recent studies have identified the quadruple semitendinosus graft as a comparable option for ACL reconstruction (4). Advances in hamstring graft fixation have led to similar strengths of fixation between BPTB and semitendinosus/gracilis grafts (5). Both techniques now offer a high degree of strength and stiffness in the reconstructed ligament. Additionally, use of the patient’s own tissue eliminates the risk of rejection and good to excellent results are reported in 85–90% of patients receiving either of these grafts for ACL reconstructions. Nonetheless, with patellar tendon autografts, many patients experience impaired function and significant morbidity at the donor site including secondary anterior knee pain, patellar tendonitis, infrapatellar contracture, and patellar fracture. Likewise, hamstring weakness and saphenous nerve injury can be seen secondary to hamstring harvest in semitendinosus/gracilis autograft ACL reconstruction (6).
To avoid complications resulting from donor site morbidity in autograft reconstructions, allograft reconstruction has also been examined. While eliminating the donor site morbidity, the use of allografts is not currently considered advantageous due to a limited donor tissue supply, delayed biological incorporation, risks of disease transmission and tissue rejection. In an attempt to overcome these concerns, research into the use of synthetic prosthetic ligaments began over 30 years ago and continues today in hopes of eliminating donor site morbidity and reducing the risk of disease transmission and supply shortages. One of the main obstacles in finding an adequate prosthetic replacement for the ACL is the longevity of the graft, with autogenous tissue consistently proving to be a more durable and long-lasting replacement than the many biomaterials that have been applied to ACL replacement (7).
PROSTHETIC BEGINNINGS AND PAST USE
The first attempts at a synthetic ACL reconstruction were conducted by Alwyn-Smith in 1918 using silk sutures; they however failed within 3 months (8). Reconstruction with a prosthetic ligament made of polytetrafluoroethylene (PTFE) with embedded carbon or aluminum oxide fibers (Proplast) was first reported in 1973 (9). Results with this system yielded an average time to breakage of just over 1 year. A report by James et al. suggests that only 52% of reconstructions utilizing this graft yielded satisfactory results (10). Satisfactory results include all grafts that patients were content with and had not ruptured at time of lastest follow-up.
Current classification systems for synthetic replacements of the ACL include grafts, ligament augmentation devices, or total prosthetics. Grafts (polyethylene, PTFE), typically fixed at both ends, were the initial focus of synthetic ACL replacement and were meant to provide stability to the ACL-deficient-knee until secondary reconstruction procedures gained popularity (11). Similarly, ligament augmentation devices (polypropylene, polyester) were intended to provide immediate protection for autogenous tissue grafts until revascularization was complete and the ingrown tissue was capable of withstanding local tensile and compressive forces. Unfortunately, these devices may in fact accomplish the opposite of their intended purpose by stress-shielding autogenous tissue, preventing it from developing adequate mechanical strength. Fixed at both ends like a graft, the total prosthesis was intended as a permanent ACL replacement without any soft-tissue ingrowth that would allow the patient to begin aggressive rehabilitation immediately as no tissue maturation or revascularization was required. While the implantation of a full-strength ACL replacement generally led to excellent short-term results, long-term efficacy results were poor due to wear and ensuing rupture of the prosthesis (11).
Combining the mechanical focus provided by these prior prosthetic approaches with tissue engineering principles has led to the development of a scaffold design intended to serve as a foundation for soft tissue in-growth. Scaffolds may be permanent or may be 31 Vol. 11 No. 1 Anterior Cruciate Ligament Reconstruction: A Look at Prosthetics intended to gradually degenerate as the host tissue replaces them. Problems associated with the biological incorporation of scaffolds include variability of tissue in-growth, immature degeneration of the implant and insufficient maturation of the host tissue resulting in an inability of the scaffold to withstand inherent mechanical stresses placed on the ACL. Current directions in this area involve the use of tissue engineering focusing on developing a mechanically and biologically functional matrix for the scaffold and the use of in vitro mechanical signals to guide new tissue development (11). Table 1 summarizes the advantages of the various prosthetic ACL graft options that have been attempted and are discussed below.
CARBON FIBRE PROSTHETICS
Some of the earliest attempts to discover an appropriate alternative to the biological graft resulted in a variety of carbon fibre prostheses that varied greatly with respect to material and implantation technique. In 1977, Jenkins et al. were the first to use a flexible carbon fibre implant, and suggested that the carbon fibre induced the formation of tendon in animals and humans (12). However, subsequent studies reported the generation and migration of carbon wear particles into the joint space and regional lymph nodes following implantation of the prosthesis (12). To combat this problem, attempts were made to coat the carbon fibre with collagen and absorbable polymers. Of particular interest was a carbon fibre implant proposed by Alexander et al. coated with a co-polymer of polylactic acid (PLA) and polycapralactone (13). The carbon filaments were thought to act as a scaffold for tissue ingrowth by evenly distributing and reducing the interfacial stress between the implant and soft tissue attachment, while the PLA/polycapralactone would protect the fibre during implantation. Over time, the PLA was meant to resorb and the carbon fibres degrade as new tissue developed, thereby encouraging normal tissue regeneration without permanently replacing it (13). A 24-month study involving 82 patients was conducted by Weiss et al. to further examine the PLA coated prosthesis using a variety of subjective and objective measures (pain, stability, function, and isokinetic strength testing), which revealed significant improvements over the duration of the study (14). Arthroscopic evaluation demonstrated collagenous tissue ingrowth, confirmed by histological studies, to be composed of Type I and III collagen in similar proportions to that found in normal healing ligamentous tissue (14).
A similar design was used in the Surgicraft ABC prosthetic ACL (Surgicraft Ltd., Redditch UK) composed of carbon and polyester fibres oriented in a partial braid by a zig-zag pattern. However, a study by O’Brien et al. with an average follow-up of 34 months showed only 11 of 31 knees (41%) had good results defined as a Lysholm score greater than 76. The Lysholm knee score is an outcome measure that assesses knee function on a 100 point scale. The authors also noted unacceptable stretching and complete rupture as major complications and concluded that the implant is unsuitable for clinical use (15).
GORE-TEX PERMANENT PROSTHESIS
The Gore-Tex ligament prosthesis is composed of a single long fiber of expanded polytetrafluoroethylene (PTFE) arranged into loops. Extensive mechanical testing has shown that the resulting ultimate tensile strength is about 3 times that of the human ACL and the results from cyclical creep tests and bending fatigue testing seem to identify Gore-Tex as the strongest synthetic ACL replacement in terms of pure material stability (16). Bolton and Bruchman reported that 129 out of 130 patients receiving a high strength PTFE ligament showed improved knee stability at 15 months or less (16). Glousman et al. reported an initial improvement upon physical examination and subjective scores. However, at mean follow-up of 18 months, they reported a progressive loosening of the prosthesis (17). Similarly, Woods et al. presented 2- and 3-year follow-up of Gore-Tex ACL reconstructions and showed a similar pattern of early improvement post-operatively, but deterioration over time. They reported an overall failure rate of 33% at 3-year follow-up (18). Indelicato et al.’s follow-up of Gore-Tex implant ACL reconstructions showed a 90% success rate at 2 years versus only a 76% success rate at 3 years or more (19). Despite similar reports describing complications with the Gore-Tex ACL prosthesis, subjective results in several studies remain acceptable at 60–80 % (17, 19). The Gore-Tex ACL prosthesis is currently FDA approved for use in patients who have had a failed autogenous intra-articular graft procedure.
DACRON
With its success as a vascular surgery implant (20), various forms of Dacron grafts have been developed as a scaffold for ACL replacements. The implant is a composite of four tightly woven polyester strips wrapped in a sheath of loosely woven velour, designed to minimize abrasion of the graft and act as a scaffold for fibrous tissue ingrowth. A report by Lukianov et al. reviewed the short-term follow-up (mean 28 months) of 41 patients who underwent ACL reconstruction with the Stryker Dacron ligament prosthesis (3). Seventy-five percent of the patients were found to have a negative Lachman, anterior drawer, and pivot shift at their most recent follow-up. However, Richmond et al. reported failure rates of 37.1% in a study of Dacron reconstruction with mean long-term follow-up of 50 months (21). Likewise, Barrett et al. reported higher failure rates of 47.5% after a four-year follow-up period (22). A clinical study by Lopez-Vazquez et al. examining ACL reconstruction with a Dacron prosthesis showed a similar deterioration of results after the first post-operative year (23). With the initial short-term strength shown by these grafts offset by their poor long-term stability, Dacron grafts should not be considered a viable alternative for ACL reconstruction.
LEEDS-KEIO ARTIFICIAL LIGAMENT
With the desire to design a graft that combined the properties of a permanent prosthesis and a tissue-promoting scaffold, Fujikawa and Seedhom developed the Leeds-Keio artificial ligament: a polyester mesh-like structure anchored to the femur and tibia with bone plugs (24). This mesh was intended as a scaffold for soft tissue ingrowth through the intra-articular and extra-articular sections of the ligament, eventually uniting the bone plugs. The implant was considered sufficiently flexible to be effective with a maximal tensile strength of approximately 2100 Newtons (N), which significantly exceeds that of the average young adults’ natural ACL (about 1730 N) (24). Initial descriptions from the inventors also described minimal articular wear with the ligament (24). The inventors of this graft have reported successful clinical results with arthroscopic observations documenting neoligamentous tissue within the implanted Dacron scaffold. Other investigators, however, have reported the ingrowth of non-aligned fibrous tissue (i.e. non-neoligamentous tissue) within the device after implantation and suggested that the Leeds-Keio ligament did not serve as a true scaffolding graft, but instead behaved as a permanent load-bearing prosthesis, subject to long-term failure in the joint (25).
McLoughlin and Smith presented a 3.8 year follow-up study of 25 patients implanted with the Leeds-Keio ligament for chronic ACL instability. They reported a low complication rate and considerable success in the elimination of instability after finding good post-operative results with the anterior drawer test using an arthrometer at 90° of flexion (26). Nevertheless, ensuing long-term follow-up studies showed a deterioration of results after the first post-operative year and a large number of long-term graft ruptures despite excellent early results in stability testing and on the Lysholm scale (27). These findings were similar to earlier results reported by Schindhelm, who found that good early results in a sheep model were not maintained (28). Due to the number of long-term graft ruptures and the lack of long-term stability provided, the Leeds-Keio ligament is no longer suitable for reconstruction of the human ACL (27, 28).
KENNEDY LIGAMENT AUGMENTATION DEVICE (LAD)
Kennedy et al. introduced the concept of the Ligament Augmentation Device (LAD) in 1980 (29). The graft, composed of a band-like braid of polypropylene, was originally developed to reinforce the area of pre-patellar tissue considered to be a weak area of autogenous patellar tendon grafts. Use of this prosthesis employed the MacIntosh/Marshall transfer of a portion of the rectus femoris tendon, pre-patellar tissue, and central third of the patellar tendon in an over-the-top fashion (30). Originally, the graft was developed to protect the autogenous tissue graft from excessive stresses during the initial remodelling phase (characterized by degeneration and revascularization), allowing for earlier resumption of pre-operative activity levels (29). Research has shown that the percentage of load accepted by the LAD varies according to the type of graft employed and method of reconstruction. Comparisons of the patellar tendon and semitendinosus/gracilis LAD composite grafts revealed that the LAD will accept approximately 28% and 45% of the applied load, respectively (31). To prevent excessive stress shielding that would otherwise prevent the autogenous tissue from developing a normal functional tensile strength, the LAD was attached to the bone at only one end. Despite a promising beginning, the suture interface between the LAD and the graft was identified as the weak link of the composite.
Whether collagen fibres become truly incorporated into the LAD remains a controversial issue. Most histological evidence has been derived from animal studies and thus may not be fully indicative of results in humans. Nevertheless, this evidence seems to point to adequate longitudinal collagenization of the graft with inconclusive results on whether or not collagen ingrowth within the LAD has occurred (32). Furthermore, as an intra-articular foreign body, the LAD has been reported to induce an inflammatory response characterized by foreign body giant cells and macrophages in the surrounding tissue. A review by Kumar et al. reported that the majority of complications seen following use of the LAD were characterized as effusions and reactive synovitis, likely a result of LAD-induced inflammatory response (23). The decline in use of the aforementioned MacIntosh/Marshall transfer, combined with the weak graft-prosthetic suture interface and propensity of the LAD to cause high rates of post-operative synovitis have resulted in a lack of widespread use of the device.
THE FUTURE: LIGAMENT ADVANCED REINFORCEMENT SYSTEM (LARS) ARTIFICIAL LIGAMENT
The Ligament Advanced Reinforcement System (LARS) (Arc-sur-Tille, France) artificial ligament consists of fibres made of polyethylene terephthalate (PET). An intra-osseous segment is composed of longitudinal fibres bound together by a transverse knitted structure while an intra-articular segment is composed of parallel longitudinal fibres twisted at 90°. The main innovation of this artificial ligament lies in its ability to mimic the natural ligamentous structure and reduce shearing forces by orientating the free fibers of the intra-articular portion of the graft clockwise or counter-clockwise for use in right and left knees, respectively. Furthermore, the PET fibres of the intra-articular segment are designed to encourage tissue ingrowth due to the porosity of the material, allowing ingrowth from the surrounding osseous tunnels. Ideally, such tissue ingrowth between the ligament fibres would contribute to the viscoelasticity of the graft and protect against friction at the opening of the bony canal and between the fibres themselves (1).
A study by Lavoie et al. examined ACL reconstruction with the use of the LARS artificial ligament. Thirty-eight of forty-seven patients suffered from chronic ruptures of the ACL, while nine others presented with acute or subacute ruptures at a mean follow-up of 21.9 months. Six patients had previously had an unsuccessful ACL reconstruction. The Knee Osteoarthritis Outcome Score (KOOS) was administered to assess patients’ opinions regarding their knee, while a modified International Knee Documentation Committee (IKDC) scoring system was used to examine knee stability. The IKDC form initially consisted of seven knee-related parameters that were each rated as either normal, nearly normal, abnormal, and severely abnormal. The worst score amongst the seven categories determined the final score. The form was later modified in 2001 to include subjective factors such as symptoms, sports activities, and ability to function. The Tegner activity scale, a subjective ten point activity scale, was obtained to assess patient activity levels. A Telos radiographic stress system was used to examine anterior knee displacement, which involves obtaining radiographs with the knee in ninety degrees of flexion and a posterior force applied to the knee. When results are compared to the normal knee, this test helps in the assessment of post-operative laxity in the reconstructed knee which may indicate that the graft is failing. None of the patients presented symptoms of synovitis, but longer follow-up time is required to properly draw conclusions with respect to patient outcome. Although Tegner scores improved significantly following surgery, no patients returned to pre-injury activity levels. Data obtained by the KOOS demonstrated patient satisfaction ranged from 73.5 to 93.0 %. In comparison to the uninjured knee, post-operative Telos stress radiography and the Lachman test indicated an average posterior-anterior displacement of 7.3 mm for the involved knee (28).
A more complete examination of the LARS artificial ligament was provided in a subsequent study by the same authors. Nau et al. conducted a two-year follow-up randomized controlled trial that compared the BPTB autograft with the LARS artificial ligament method of ACL reconstruction in 53 patients with chronic instability (1). Like the preceding study, assessments included clinical examination, anterior laxity testing, as well as the KOOS, IKDC, and Tegner scores. In particular, IKDC evaluation revealed little significant difference between the two methods. Follow-up values for instrumented laxity testing were greater in the LARS group. While similar overall results were obtained for both groups, these results may suggest that a full return to activity may be hastened by using the LARS artificial ligament rather than the conventional BPTB technique (1).
Another recent study by Talbot et al. examined the use of the LARS artificial ligament for ligament reconstruction in knee dislocations (2). Twenty patients were included with a mean follow-up of 27.4 months. Each patient was evaluated using the Lysholm score, underwent clinical examination to identify ligamentous laxity and range of motion, and completed the ACL quality of life (ACL-QoL) questionnaire. The mean Lysholm score was 71.7, which is lower than scores reported by several other studies (range of 74.7 to 91.3) investigating the outcomes of knee dislocations treated with surgery as reported by Fanelli (35). Following the same trend, Telos radiometry revealed a mean residual laxity of 5 mm in patients post-surgery, which is also greater than these previously reported results. The average range of motion post-surgery was 118° with a mean fixed flexion contracture of 2° (2).
Although preliminary investigations into the use of the LARS artificial ligament have been encouraging, concerns regarding the risk of rupture remain and must be addressed through long-term follow-up studies.
TISSUE ENGINEERING ADVANCES
Permanent synthetic prostheses are capable of duplicating the mechanical and structural properties of the ACL. However, they generally tend to lose strength with time. Tissue-based or tissue-aided implants offer the additional possibility of the restoration of normal joint kinematics while the mechanical behaviour of these implants is expected to improve over time as tissues are remodelled within the knee (36). An ideal ACL scaffold must meet the immediate functional mechanical demands within the reconstructed knee, however, they must also degrade at a rate similar to that of tissue ingrowth. Accordingly, the ACL scaffold should lose its mechanical integrity while allowing the remodelled tissues to gain strength and accept an increasing amount of the mechanical demands placed on the ACL. Current research into this novel tissue-engineering approach has focused on seeding either collagen-based scaffolds or synthetic biodegradable polymers with a variety of different cell types. In hopes of stimulating early healing, reducing biomaterial-related inflammatory response and improving neoligament formation, several researchers have sought to adopt a cell sheeting technique to improve the performance of the synthetic ACL scaffold.
Several groups have conducted experiments on this tissue-engineered approach to ACL ligament reconstruction using both fibroblast-seeded synthetic scaffolds and collagen-based prosthetics (37, 38). Bellincampi et al. measured the ingrowth characteristics of rabbit fibroblasts on skin and ACL scaffolds (39). Subsequent in vivo studies suggested that fibroblast-seeded collagen scaffolds were viable after re-implantation into the donor rabbit. The major limitations of these approaches are the allogenicity of the collagen scaffolds, often leading to further complications. Collagen-based constructs also suffer from batch-to-batch variability, making consistent reproduction of these prostheses difficult. Collagen does not offer the same flexibility for modification that is reported with the technology of biodegradable polymers (6).
As an alternative to the scaffolds made of non-degradable polymers, investigators have begun to examine biodegradable materials that would provide immediate stabilization to the repaired ligament but would also act as a scaffold for the ingrowth and/or replacement by host cells. Cao et al. described the generation of neo-tendons in a nude mice model by implanting polyglycolic acid (PGA) scaffolds seeded with bovine tendon fibroblasts in the subcutaneous space of athymic mice (40). Using a similar system, Koski et al. reported the formation of ligament-like structures when fibroblasts isolated from bovine cruciate ligaments were seeded onto PGA scaffolds and implanted subcutaneously in nude mice (6). In both studies, the tissue developed histological characteristics similar to normal tendon and ligament over time.
Ouyang et al. reported that bone marrow stromal cells (bMSCs) seeded onto poly-lactic and -glycolic acid (PLGA) scaffolds grew as a multi-layer of cells intertwined in a collagen matrix synthesized by the cells 35 Vol. 11 No. 1 Anterior Cruciate Ligament Reconstruction: A Look at Prosthetics themselves. They also noted that the cell sheet formed faster than scaffolds seeded with terminally differentiated cells, such as fibroblasts and smooth muscle cells (41). They reported that degradation of the scaffolds occurred over time, as indicated by a decrease in failure load. Altman et al. seeded 6-cord silk scaffolds with human bone marrow stromal cells and cultured them for 14–21 days. Their results similarly suggested a slow degradation of the scaffolds. However, fatigue analysis and subsequent regression analysis revealed an expected matrix lifetime equivalent to 1 year in vivo. While these reports are an estimate, the authors suggest that this is a marked improvement compared to similar studies using collagen scaffolds (36).
The future of tissue engineering may also require a significant contribution from cell-specific growth factors influencing the maturation and homeostasis of the healing response of ligament tissue. Studies have suggested that individual growth factors may have an important effect on cell division and enhance ligament healing, while others have pointed to the synergistic effect of a combination of growth factors as important to cell outgrowth in ACL explants (42, 43). In a study observing the effects of several growth factors on the cell migration, proliferation and collagen production in human ACL cells, Murray et al. suggested that specific dose-response relationships may exist for the optimal activity of each growth factor (44). These authors observed that the addition of transforming growth factor (TGF)β-1 led to an increased cell population, as well as increased collagen and smooth muscle actin production in human ACL cells cultured on top of a collagen-glycosaminoglycan scaffold. Despite the complex nature of the inter-dependent factors at play during the recovery and rehabilitative period following an ACL reconstruction, much of the literature today suggests that tissue engineering techniques will lead to a new generation of ACL replacements, capable of regenerating a mechanically robust and natural ACL.
COMPUTER-ASSISTED ACL RECONSTRUCTION
In an attempt to improve the accuracy and reproducibility of ACL graft placement, several groups have turned to computer-assisted surgery in an attempt to reduce the incidence of graft failure (45, 46). These systems are capable of modeling the placement and predicting the impingement of an ACL graft based on intra-operative anatomical landmarks and signals received at an opto-electric camera. These studies reported that experience level did not affect the placement of the tunnels. Computer-assisted ACL replacement reduces variance in tunnel placement and allows residents and less experienced surgeons to limit complications and control tunnel positioning (46). Based on the success of navigation systems for total knee and hip replacements, the use of computer-assisted ACL reconstruction may lead to similarly dramatic improvements in technical and functional outcomes.
CONCLUSION
Satisfactory prosthetic replacement of the ACL has been a focus of orthopaedic research endeavours for the past thirty years. Desires to provide immediate mobility and strength to the ACL-deficient knee while avoiding the donor site morbidity caused by the commonly used autograft surgical techniques continually drive new research initiatives. Most of the grafts that have been developed to date have failed due to unsatisfactory long-term physiologic and functional performance. Most permanent ACL prostheses are prone to creep, fatigue, and mechanical failure within several years after implantation (40). Tissue ingrowth scaffolds and ligament augmentation devices require further refinement to provide effective mechanical support while avoiding stress-shielding of the host tissue. In view of these factors, prosthetics are not widely used today in ACL reconstruction, and autogenous tissue grafts remain the gold standard used by the majority of surgeons. Perhaps development of resorbable, tissue-inducing and cell-seeded biomaterials will improve the long-term biomechanical performance of the reconstructed anterior cruciate ligament. Advances in tissue engineering combined with developments in molecular biology and gene therapy may couple with the rapid gains in computer-assisted surgery to provide improved options for the ACL-deficient knee, with a greater potential to restore its pre-injury state.

anterior cruciate ligament
prosthetics
tissue engineering
biomaterials
synthetic grafts