Lifetime risk and genetic predisposition to post-traumatic OA of the knee in the UK Biobank.

OBJECTIVE: Acute knee injury is associated with post-traumatic OA (PTOA). Very little is known about the genome-wide associations of PTOA when compared with idiopathic OA (iOA). Our objective was to describe the development of knee OA after a knee injury and its genetic associations in UK Biobank (UKB). DESIGN: Clinically significant structural knee injuries in those ≤50 years were identified from electronic health records and self-reported data in 502,409 UKB participants. Time-to-first knee osteoarthritis (OA) code was compared in injured cases and age-/sex-matched non-injured controls using Cox Proportional Hazards models. A time-to-OA genome-wide association study (GWAS) sought evidence for PTOA risk variants 6 months to 20 years following injury. Evidence for associations of two iOA polygenic risk scores (PRS) was sought. RESULTS: Of 4233 knee injury cases, 1896 (44.8%) were female (mean age at injury 34.1 years [SD 10.4]). Over a median of 30.2 (IQR 19.5-45.4) years, 1096 (25.9%) of injured cases developed knee OA. The overall hazards ratio (HR) for knee OA after injury was 1.81 (1.70,1.93), P = 8.9 × 10-74. Female sex and increasing age at injury were associated with knee OA following injury (HR 1.15 [1.02,1.30];1.07 [1,07,1.07] respectively). OA risk was highest in the first 5 years after injury (HR 3.26 [2.67,3.98]), persisting for 40 years. In 3074 knee injury cases included in the time-to-OA GWAS, no variants reached genome-wide significance. iOA PRS was not associated with time-to-OA (HR 0.43 [0.02,8.41]). CONCLUSIONS: Increasing age at injury and female sex appear to be associated with future development of PTOA in UKB, the risk of which was greatest in the 5 years after injury. Further international efforts towards a better-powered meta-analysis will definitively elucidate genetic similarities and differences of PTOA and iOA.

Development of Tissue-Engineered Ligaments: Elastin Promotes Regeneration of the Rabbit Medial Collateral Ligament.

When ligaments are injured, reconstructive surgery is sometimes required to restore function. Methods of reconstructive surgery include transplantation of an artificial ligament and autotransplantation of a tendon. However, these methods have limitations related to the strength of the bone-ligament insertion and biocompatibility of the transplanted tissue after surgery. Therefore, it is necessary to develop new reconstruction methods and pursue the development of artificial ligaments. Elastin is a major component of elastic fibers and ligaments. However, the role of elastin in ligament regeneration has not been described. Here, we developed a rabbit model of a medial collateral ligament (MCL) rupture and treated animal knees with exogenous elastin [100 µg/(0.5 mL·week)] for 6 or 12 weeks. Elastin treatment increased gene expression and protein content of collagen and elastin (gene expression, 6-fold and 42-fold, respectively; protein content, 1.6-fold and 1.9-fold, respectively), and also increased the elastic modulus of MCL increased with elastin treatment (2-fold) compared with the controls. Our data suggest that elastin is involved in the regeneration of damaged ligaments.

Gene expression in human meniscal tears has limited association with early degenerative changes in knee articular cartilage.

Purpose/Aim: Meniscus tears are a common injury to the knee associated with the development of osteoarthritis. Gene expression in the injured meniscus may be associated with early degeneration in the articular cartilage. The purpose of this study was to test the hypothesis that gene expression in meniscus tears is associated with early degenerative changes in the articular cartilage at the time of partial meniscectomy. MATERIALS AND METHODS: Torn meniscus was removed at the time of partial meniscectomy in 68 patients without radiographic osteoarthritis. Meniscal mRNA expression was measured by quantitative PCR for multiple molecular markers of osteoarthritis and cartilage homeostasis. The presence of early degenerative changes in the knee was recorded by X-ray (N = 63), magnetic resonance imaging (MRI, N = 48), and arthroscopy (N = 63). Gene expression was tested for correlation with the presence/absence of degenerative changes after adjusting for age, sex, and body mass index. RESULTS: Overall gene expression varied significantly with degenerative changes based on X-ray (P = 0.047) and MRI (P = 0.018). The linear combination of gene variation was also significant. However, only adiponectin (ADIPOQ) (P = 0.015) was expressed at a significantly lower level in patients with chondrosis on MRI, while the expression of ADIPOQ (P = 0.035) and resistin (RETN) (P = 0.017) was higher in patients with early degenerative changes on X-ray. None of the genes varied significantly with presence/absence of chondrosis as measured by arthroscopy. CONCLUSIONS: There is an overall association of gene expression in meniscal tears to early degenerative changes in the knee, but only a limited number of specific genes demonstrate this relationship. The roles of adiponectin and resistin in knee injury and osteoarthritis deserve further study.

Two Genetic Loci associated with Medial Collateral Ligament Injury.

Medial collateral ligament (MCL) injuries are a common knee injury, especially in competitive athletes. Identifying genetic loci associated with MCL injury could shed light on its etiology. A genome-wide association screen was performed using data from the Research Program in Genes, Environment and Health (RPGEH) including 1 572 cases of MCL injury and 100 931 controls. 2 SNPs (rs80351309 and rs6083471) showed an association with MCL injury at genome-wide significance (p<5×10-8) with moderate effects (odds ratios=2.12 and 1.57, respectively). For rs80351309, the genotypes were imputed with only moderate accuracy, so this SNP should be viewed with caution until its association with MCL injury can be validated. The SNPs rs80351309 and rs6083471 show a statistically significant association with MCL injury. It will be important to replicate this finding in future studies.

Analysis of the Relationship between Antioxidant Enzyme Gene Polymorphisms and Their Activity in Post-Traumatic Gonarthrosis.

Analysis of polymorphisms of genes encoding antioxidant enzymes SOD1 (G7958A), SOD2 (T58C), CAT (C-262T), and GSTP1 (Ile105Val) in 93 patients with post-traumatic gonarthrosis showed that GSTP1 Ile105Val polymorphism is often associated with heterozygous mutation in catalase gene CAT C-262T. In gonarthrosis, catalase activity in peripheral blood mononuclear cells in patients with CT genotype of the C-262T locus of CAT gene more than 2-fold surpassed that in CC genotype and more than 50% surpassed the normal. Changes in the balance of activity of antioxidant enzymes can affect viability of mononuclear cells.

Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury.

OBJECTIVE: Sclerostin plays a major role in regulating skeletal bone mass, but its effects in articular cartilage are not known. The purpose of this study was to determine whether genetic loss or pharmacologic inhibition of sclerostin has an impact on knee joint articular cartilage. METHODS: Expression of sclerostin was determined in articular cartilage and bone tissue obtained from mice, rats, and human subjects, including patients with knee osteoarthritis (OA). Mice with genetic knockout (KO) of sclerostin and pharmacologic inhibition of sclerostin with a sclerostin-neutralizing monoclonal antibody (Scl-Ab) in aged male rats and ovariectomized (OVX) female rats were used to study the effects of sclerostin on pathologic processes in the knee joint. The rat medial meniscus tear (MMT) model of OA was used to investigate the pharmacologic efficacy of systemic Scl-Ab or intraarticular (IA) delivery of a sclerostin antibody-Fab (Scl-Fab) fragment. RESULTS: Sclerostin expression was detected in rodent and human articular chondrocytes. No difference was observed in the magnitude or distribution of sclerostin expression between normal and OA cartilage or bone. Sclerostin-KO mice showed no difference in histopathologic features of the knee joint compared to age-matched wild-type mice. Pharmacologic treatment of intact aged male rats or OVX female rats with Scl-Ab had no effect on morphologic characteristics of the articular cartilage. In the rat MMT model, pharmacologic treatment of animals with either systemic Scl-Ab or IA injection of Scl-Fab had no effect on lesion development or severity. CONCLUSION: Genetic absence of sclerostin does not alter the normal development of age-dependent OA in mice, and pharmacologic inhibition of sclerostin with Scl-Ab has no impact on articular cartilage remodeling in rats with posttraumatic OA.

Transcriptome analysis of injured human meniscus reveals a distinct phenotype of meniscus degeneration with aging.

OBJECTIVE: Meniscus tears are associated with a heightened risk of osteoarthritis. This study aimed to advance our understanding of the metabolic state of injured human meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to the patient's age and degree of cartilage chondrosis. METHODS: The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (n = 12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. Variations in the relative changes in gene expression with age and chondrosis were analyzed, and integrated biologic processes were investigated computationally. RESULTS: We identified a set of genes in torn menisci that were differentially expressed with age and chondrosis. There were 866 genes that were differentially regulated (≥1.5-fold difference and P < 0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed, while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed, and those involved in T cell differentiation and apoptosis were elevated. CONCLUSION: Differences in age-related gene expression suggest that in older adults, meniscal cells might dedifferentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but they maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis.

Sex-steroid regulation of relaxin receptor isoforms (RXFP1 & RXFP2) expression in the patellar tendon and lateral collateral ligament of female WKY rats.

UNLABELLED: The incidence of non-contact knee injury was found higher in female than in male and is related to the phases of the menstrual cycle. This raised the possibility that female sex-steroids are involved in the mechanism underlying this injury via affecting the expression of the receptors for relaxin, a peptide hormone known to modulate ligament laxity. Therefore, this study aims to investigate the effect of sex-steroids on relaxin receptor isoforms (RXFP1 & RXFP2) expression in the ligaments and tendons of the knee. METHODS: Ovariectomized adult female WKY rats were treated with different doses of estrogen (0.2, 2, 20 µg/kg), progesterone (4mg) and testosterone (125 & 250µg/kg) for three consecutive days. At the end of the treatment, the animals were sacrificed and the patellar tendon and lateral collateral ligament were harvested for mRNA and protein expression analyses by Real Time PCR and Western blotting respectively. RESULTS: RXFP1, the main isoform expressed in these knee structures and RXFP2 showed a dose-dependent increase in expression with estrogen. Progesterone treatment resulted in an increase while testosterone caused a dose-dependent decrease in the mRNA and protein expression of both relaxin receptor isoforms. DISCUSSION: Progesterone and high dose estrogen up-regulate while testosterone down-regulates RXFP1 and RXFP2 expression in the patellar tendon and lateral collateral ligament of rat's knee. CONCLUSION: Relaxin receptor isoforms up-regulation by progesterone and high dose estrogen could provide the basis for the reported increase in knee laxity while down-regulation of these receptor isoforms by testosterone could explain low incidence of non-contact knee injury in male.

The GDF5 SNP is associated with meniscus injury and function recovery in male Chinese soldiers.

Genetic factor have previously been shown to play an important role in sports injuries and recovery. GDF5 Single-Nucleotide Polymorphism rs143383 has been recently reported to be associated with fracture susceptibility. Furthermore, the effect of GDF5 during the recovery processes of trauma is increased. In the present study, we aimed to evaluate whether this SNP was associated with susceptibility to the meniscus injury and postoperative recovery in Chinese male soldiers. GDF5 SNP was genotyped in 135 male soldiers with meniscus injury and 400 healthy male controls. Moreover, the function recovery of the soldiers suffering from the meniscal repair was also assessed. Our data showed that the GDF5 TT genotype (60.0 vs. 47.25%; P=0.010) and T allele (76.3 vs. 68.75%; P=0.019) were significantly over-represented in the meniscus injury group compared with the control group. We found that the TC (P<0.05), CC (P<0.05) and C carriers (P<0.05) genotype exhibited significantly higher Lysholm Scores than the TT genotype at 1 month postoperative. In addition, the CC (P<0.05) genotype also demonstrated significantly higher Lysholm Scores than the TT genotype 2 months postoperative. Taken together, our results revealed that the GDF5 SNP was associated with susceptibility to the meniscus injury and postoperative function recovery in Chinese male soldiers.

[Non-traumatic osteochondral lesions of the knee joint during growth : Juvenile osteochondritis dissecans (JOCD) of the knee]. / Nichttraumatische osteochondrale Läsionen des Kniegelenkes im Wachstumsalter : Juvenile Osteochondrosis dissecans (JOCD) des Kniegelenkes.

BACKGROUND: Juvenile osteochondritis dissecans of the knee joint is the most common osteochondral lesion during growth, usually occurring between the 10th and 14th year of age. PATHOGENESIS: Repetitive microtraumata lead to a subchondral osseus lesion, which is commonly located at the medial aspect of the femoral condyle. Sport activities are considered to be the main cause, although genetic and hereditary factors as well as vitamin D3 deficiency also play a role. Current classification systems distinguish between stable and unstable osteochondral lesions, which is decisive for further treatment. TREATMENT: Stable lesions may heal through conservative treatment by avoiding weight bearing and sport. Unstable lesions, on the other hand, can lead to a complete defect of the joint surface with the formation of a free joint body. In such cases, various surgical techniques aim at reconstructing the surface of the joint, in order to reduce the risk of secondary arthritis.

Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis.

OBJECTIVE: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. DESIGN: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. RESULTS: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. CONCLUSIONS: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries.

Matrix metalloproteinase genes on chromosome 11q22 and the risk of anterior cruciate ligament (ACL) rupture.

As matrix metalloproteinases (MMPs) are critical to ligament homeostasis and integrity, the aim of this study was to investigate whether four functional polymorphisms within four MMP genes, which cluster on chromosome 11q22 associate with risk of ACL ruptures. Three hundred and forty-five [129 with ACL ruptures (ACL group) and 216 asymptomatic controls (CON group)] unrelated Caucasians were recruited for this case-control study. Fifty-four participants reported non-contact mechanisms of ACL rupture (NON subgroup). All participants were genotyped for the MMP10 C/T rs486055, MMP1 1G/2G rs1799750, MMP3 G/A rs679620 and MMP12 A/G rs2276109 variants. After adjusting for sex, age and weight, the AG and GG genotypes of the MMP12 rs2276109 variant were significantly (P=0.030) under-represented among the NON subgroup (14%), when compared with the CON group (26%). No other variants were significantly different between groups. Adjusted for the same confounders, the two four-variant haplotypes T-1G-A-A (CON 14%, ACL 9%, P=0.033) and C-2G-G-G (CON 14%, NON 5%, P=0.021) were significantly different between the CON and the ACL groups, and the CON group and the NON subgroup, respectively. This is the first report that indicates an association between the chromosomal region 11q22 and the risk of ACL rupture.

In vitro inhibition of compression-induced catabolic gene expression in meniscal explants following treatment with IL-1 receptor antagonist.

BACKGROUND: Damage to the knee meniscus may result in tears that are difficult or unable to heal, and are often treated by partial removal of the damaged tissue. In vitro, 20% dynamic compressive strains on meniscal tissue explants have resulted in an increase in the release of sulfated glycosaminoglycans (GAG) and nitric oxide (NO) from the tissue explants and increased expression of matrix metalloproteinases (MMP) and interleukin-1α (IL-1α). The objective of this study was to explore the efficacy of IL-1 blockade on the expression of a wide range of genes, as well as NO and GAG release, following dynamic compression of porcine meniscal explants. METHODS: Explants were dynamically compressed for 2 h at 1 Hz to 0, 10, or 20% strain with and without a pre-treatment of 500 ng/ml interleukin-1 receptor antagonist (IL-1RA). Relative changes in gene expression of IL-1α, MMP-1, -3, -13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS-4), ADAMTS-5, iNOS, aggrecan, and COX-2, as well as changes in NO and GAG release, were measured with standard biochemical assays. RESULTS: Expression of IL-1α, MMP-3, MMP-13, and ADAMTS-4 in superficial explants was significantly downregulated at 20% dynamic strain compared to 10% strain following treatment with IL-1RA. GAG and NO release were not significantly influenced by IL-1RA treatment. CONCLUSIONS: Treatment of meniscal explants with IL-1RA inhibited the expression of many catabolic genes following a single bout of high dynamic strain. IL-1RA may therefore be a potential therapy option during the acute phase of meniscal tear or meniscectomy treatment.

Enhanced meniscal repair by overexpression of hIGF-1 in a full-thickness model.

The importance of the menisci to the well-being of the normal knee is well-documented. However, there is no ideal repair or reconstructive approach for damaged menisci. Gene therapy provides one promising alternative strategy, especially when combined with injectable tissue engineering to achieve minimally invasive clinical application. We asked whether the introduction of human insulin-like growth factor 1 (hIGF-1) gene could improve the repair of full-thickness meniscal defects. We created full-thickness meniscal defects in the "white area" of the anterior horn in 48 goats. Bone marrow stromal cells with the transfection of hIGF-1 gene and injectable calcium alginate gel were mixed together to repair the defects; three control groups included cells without transfection, gel without cells, and defects left empty. After 4, 8, and 16 weeks, the animals were euthanized and the excised defects were examined by macroscopic assessment, histological analysis, electron microscopy, proteoglycan determination, and MRI. Sixteen weeks after surgery the repaired meniscal defects were filled with white tissue similar to that in normal meniscal fibrocartilage. The repair tissue was composed of cells embedded within matrix that filled the spaces of the fibers. The proteoglycan content in the gene-enhanced tissue engineering group was higher than those in the control groups, and less than that in the normal meniscus. The results suggest full-thickness meniscal defects in regions without blood supply can be reconstructed with hIGF-1-enhanced injectable tissue engineering.

The morphology and histology study on rabbit degenerated medial meniscus after posterior cruciate ligament rupture.

The morphology and histology changes in the medial meniscus after posterior cruciate ligament (PCL) rupture are poorly understood. Forty-eight rabbits were divided into matched mode pairs; each rabbit had an experimental side, in which the PCL was transacted, and a control side. At the 4, 8, 16 and 24 weeks after the PCL transection, each of the 12 rabbits was killed. Histology was performed to detect the expression of the tissue inhibitors of metalloproteinases-1 (TIMP-1), matrix metalloproteinase (MMP)-1 and MMP-13 in the medial meniscus. We found that medial meniscus displayed significant degenerative characteristics in morphology. The histological evaluation of the degeneration found that the expression levels of TIMP-1, MMP-1 and MMP-13 in the medial meniscus were higher in the experiment side than those in the control side (P<0.05). The expression of both TIMP-1 and MMP-13 was initially elevated and then decreased. The MMP-1 expression reached its peak swiftly and then maintained a relatively high level. There were clear time-dependent degenerative changes in the histology of the medial meniscus after PCL rupture. The high expression of TIMP-1, MMP-1 and MMP-13 in the cartilage may be responsible for the degeneration, and PCL rupture may trigger meniscus degradation and ultimately osteoarthritis.

Tendon and ligament injuries: the genetic component.

Tendons and ligaments within the upper and lower limbs are some of the more common sites of musculoskeletal injuries during physical activity. Several extrinsic and intrinsic factors have been shown to be associated with these injuries. More recently, studies have suggested that there is also, at least in part, a genetic component to the Achilles tendon, rotator cuff and anterior cruciate ligament injuries. However, specific genes have not been suggested to be associated with rotator cuff or anterior cruciate ligament injuries. Sequence variants of the tenascin C (TNC) gene, on the other hand, have been shown to be associated with Achilles tendinopathies and Achilles tendon ruptures, whereas a variant of the collagen V alpha 1 (COL5A1) gene has also been shown to be associated with Achilles tendinopathies. Both genes encode for important structural components of tendons and ligaments. The COL5A1 gene encodes for a component of type V collagen, which has an important role in regulating collagen fibre assembly and fibre diameters. The TNC gene, on the other hand, encodes for TNC, which regulates the tissue's response to mechanical load. To date, only variants in two genes have been shown to be associated with Achilles tendon injuries. In addition, although specific genes have not been identified, investigators have suggested that there is also a genetic component to both rotator cuff and anterior cruciate ligament injuries. In future, specific genotypes associated with increased risk of injury to specific tendons and ligaments can prevent these injuries by identifying individuals at higher risk.

Understanding and preventing noncontact anterior cruciate ligament injuries: a review of the Hunt Valley II meeting, January 2005.

The incidence of noncontact anterior cruciate ligament injuries in young to middle-aged athletes remains high. Despite early diagnosis and appropriate operative and nonoperative treatments, posttraumatic degenerative arthritis may develop. In a meeting in Atlanta, Georgia (January 2005), sponsored by the American Orthopaedic Society for Sports Medicine, a group of physicians, physical therapists, athletic trainers, biomechanists, epidemiologists, and other scientists interested in this area of research met to review current knowledge on risk factors associated with noncontact anterior cruciate ligament injuries, anterior cruciate ligament injury biomechanics, and existing anterior cruciate ligament prevention programs. This article reports on the presentations, discussions, and recommendations of this group.

The familial predisposition toward tearing the anterior cruciate ligament: a case control study.

PURPOSE: A study of 171 surgical cases and 171 matched controls was conducted to investigate whether a familial predisposition toward tearing the anterior cruciate ligament of the knee exists. STUDY DESIGN: Case control study; Level of evidence, 3. METHODS: Patients who were diagnosed with an anterior cruciate ligament tear were matched by age (within 5 years), gender, and primary sport to subjects without an anterior cruciate ligament tear. All 342 subjects completed a questionnaire detailing their family history of anterior cruciate ligament tears. RESULTS: When controlling for subject age and number of relatives, participants with an anterior cruciate ligament tear were twice as likely to have a relative (first, second, or third degree) with an anterior cruciate ligament tear compared to participants without an anterior cruciate ligament tear (adjusted odds ratio = 2.00; 95% confidence interval, 1.19-3.33). When the analysis was limited to include only first-degree relatives, participants with an anterior cruciate ligament tear were slightly greater than twice as likely to have a first-degree relative with an anterior cruciate ligament tear compared to participants without an anterior cruciate ligament tear (adjusted odds ratio = 2.24; 95% confidence interval, 1.24-4.00). CONCLUSIONS: Findings are consistent with a familial predisposition toward tearing the anterior cruciate ligament. CLINICAL RELEVANCE: Future research should concentrate on identifying the potentially modifiable risk factors that may be passed through families and developing strategies for the prevention of anterior cruciate ligament injuries.

Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.

Overexpression of TGF-ß via rAAV-Mediated Gene Transfer Promotes the Healing of Human Meniscal Lesions Ex Vivo on Explanted Menisci.

BACKGROUND: Direct application of therapeutic gene vectors derived from the adeno-associated virus (AAV) might be beneficial to improve the healing of meniscal tears. PURPOSE: To test the ability of recombinant AAV (rAAV) to overexpress the potent transforming growth factor-ß (TGF-ß) in primary cultures of human meniscal fibrochondrocytes, in human meniscal explants, and in experimental human meniscal lesions as a new tool to enhance meniscal repair. STUDY DESIGN: Controlled laboratory study. METHODS: The effects of the candidate treatment on the proliferative and metabolic activities of meniscal cells were monitored in vitro for up to 21 days and in situ in intact and injured human menisci for up to 15 days using biochemical, immunohistochemical, histological, and histomorphometric analyses. RESULTS: Efficient production of TGF-ß via rAAV was achieved in vitro and in situ, both in the intact and injured meniscus. Application of the rAAV TGF-ß vector stimulated the levels of cell proliferation and matrix synthesis (type I collagen) compared with control gene transfer in all systems tested, especially in situ in regions of poor healing capacity and in sites of meniscal injury. No adverse effects of the candidate treatment were observed at the level of osseous differentiation, as tested by immunodetection of type X collagen. Most remarkably, a significant reduction of the amplitude of meniscal tears was noted after TGF-ß treatment, an effect that was associated with increased expression levels of the α-smooth muscle actin contractile marker. CONCLUSION: Overexpression of TGF-ß via rAAV gene transfer is capable of modulating the reparative activities of human meniscal cells, allowing for the healing of meniscal lesions by convenient injection in sites of injury. CLINICAL RELEVANCE: Direct gene-based approaches using rAAV have strong potential to develop new therapeutic options that aim at treating human meniscal defects.