Nonviral gene transfer into human meniscal cells. Part II: effect of three-dimensional environment and overexpression of human fibroblast growth factor 2

Purpose

Our aim was to study the effect of three-dimensional (3D) environment and overexpression of human fibroblast growth factor 2 (FGF-2) on meniscal fibrochondrocytes in vitro.

Methods

Human meniscal fibrochondrocytes were transfected with expression plasmid vectors carrying the Photinus pyralis luciferase gene, the Escherichia coli β-galactosidase gene or a human FGF-2 cDNA. Modified fibrochondrocytes were cultivated in 3D alginate hydrogel or cell pellets or in 2D monolayer culture.

Results

The levels of luciferase activity showed a peak at day two and returned to baseline levels by day 11, regardless of the type of cultivation. Both 3D environments supported the secretion of human FGF-2 protein upon FGF-2 transfection. Overexpression of human FGF-2 by genetically modified human meniscal fibrochondrocytes stimulated proliferation but not glycosaminoglycan synthesis only in 3D culture. Culture in alginate spheres resulted in a larger difference in cell numbers compared with pellet cultures.

Conclusions

Three-dimensional alginate spheres are well suited for the culture of genetically modified human meniscal fibrochondrocytes. These data are of value for cell-based approaches to meniscal repair using genetically modified human meniscal fibrochondrocytes overexpressing human FGF-2.     

Acceleration of articular cartilage repair by combined gene transfer of human insulin-like growth factor I and fibroblast growth factor-2 in vivo

Introduction

Improving the biochemical and structural qualities of the new tissue that fills deep osteochondral defects is critical to enhance articular cartilage repair. We developed a novel molecular therapy to increase articular cartilage repair based on a combined strategy to stimulate chondrogenesis by co-transfection of the human insulin-like growth factor I (IGF-I) and fibroblast growth factor 2 (FGF-2) in a xenogenic transplantation model.

Materials and methods

NIH 3T3 cells were transfected with expression plasmid vectors containing a cDNA for the E. coli lacZ gene (lacZ implants), the human IGF-I gene (IGF-I implants) or both the human IGF-I and FGF-2 genes (IGF-I/FGF-2 implants). The expression patterns of the transgenes were monitored in vitro for 21 days. LacZ, IGF-I and IGF-I/FGF-2 implants were transplanted into osteochondral defects in the trochlear groove of rabbits. At 3 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively.

Results

Both IGF-I and IGF-I/FGF-2 implants secreted increased levels of the corresponding recombinant proteins in vitro. In vivo, transplantation of the co-transfected IGF-I/FGF-2 implants increased the DNA content of the repair tissue, accelerated the formation of the subchondral bone and improved articular cartilage repair in a magnitude that was larger than with IGF-I alone or when compared to lacZ implants.

Conclusion

These results suggest that gene delivery of a combination of IGF-I and FGF-2 to cartilage defects may be more beneficial than application of IGF-I alone.     

Postoperative change in medial meniscal length in concurrent all-inside meniscus repair with anterior cruciate ligament reconstruction

Purpose

Meniscus repair can restore meniscal function that transfers the axial compressive force to circumferential tensile strain. However, few reports have investigated the relationship between concurrent meniscus repair with acute anterior cruciate ligament (ACL) reconstruction and postoperative meniscal position. This study aimed to evaluate medial meniscal size and clinical results in patients who underwent ACL reconstruction and concomitant all-inside medial meniscus repair.

Methods

Twenty patients underwent ACL reconstruction and concurrent medial meniscus repair of a peripheral longitudinal tear using the FasT-Fix meniscal repair device. Medial tibial plateau length (MTPL) and width (MTPW) were determined by radiographic images. We evaluated the Lysholm score, anteroposterior instability, meniscal healing and magnetic resonance imaging (MRI)-based medial meniscal length (MML) and width (MMW). Correlations between MRI-based meniscal size, radiographic measurement and height were investigated.

Results

All patients showed complete healing of the repaired meniscus in arthroscopic evaluation. However, one patient needed a subsequent meniscus repair during the follow-up period. Lysholm score and anteroposterior instability improved significantly. A better correlation was observed between MMW and MTPW than between MML and MTPL. Concurrent all-inside medial meniscus repair with ACL reconstruction significantly increased MML percentage (%MML) (100 MML/MTPL) but did not affect MMW percentage (%MMW) (100 MMW/MTPW).

Conclusions

Concurrent all-inside medial meniscus repair with ACL reconstruction had satisfactory clinical results. %MML was increased by concurrent medial meniscus repair without affecting %MMW. Our results suggest that medial meniscus repair associated with ACL reconstruction may restore meniscal function by adjusting the anteroposterior length of the torn medial meniscus.     

Allograft ligament transplantation in anterior cruciate ligament reconstruction patients with meniscal tears

Background

It currently remains unclear whether the meniscal repair clinical results were affected by the graft used in anterior cruciate ligament (ACL) reconstruction. This retrospective study designed to evaluate the difference in clinical outcomes of meniscal repair using autograft and allograft for the ACL reconstruction.

Methods

The injury of the ACL and meniscus was evaluated with MRI and treated simultaneously. One hundred and eighty-nine cases were initially fulfilled the study criteria, and had the surgery in the period June 2007 and July 2010. Thirty-four patients were lost to follow-up. Seventy-five patients underwent meniscus repair with autograft reconstruction of the ACL (autograft group) and 80 patients underwent meniscus repair with allograft reconstruction of the ACL (allograft group).

Results

The meniscus healing rate based on the clinical examination of Barrett’s criteria was 81.3 % (61/75) in the autograft group and 80.0 % (64/80) in the allograft (P > 0.05). There was no significant difference in the Lysholm scores in the allograft group compared to the allograft group (89.1 ± 10.6 versus 88.7 ± 11.2, P > 0.05). The values of immunoglobulin’s and complements (IgG, IgA, IgM, C3 and C4) were not significantly different between the two groups (P > 0.05).

Conclusion

The data support our assumption that patients undergoing meniscal repair associated with ACL reconstruction with allograft had good clinical outcomes. Although allograft implantation induces an immunological response on a subclinical level, there were no signs of allograft affecting the nature of meniscus healing.     

Beeinträchtigen Meniskusverletzungen den stabilen Stand?

Background

Meniscal lesions are known to cause a loss of proprioception, which plays an important role in the regulation of postural stability. It is unclear, however, whether meniscus injuries adversely affect not only the sense of joint position but also postural control. The objective of this study was to investigate the influence of meniscal lesions on postural stability.

Material and methods

In this prospective study, postural stability was assessed by posturography in 27 patients with unilateral meniscal lesions. Both the injured and uninjured legs were tested and compared for significant differences using a t-test.

Results

Despite the presence of an arthroscopically confirmed meniscal lesion, none of the stability indexes that we calculated revealed significant differences in postural stability between the injured and uninjured sides.

Conclusions

It was surprising to note that the loss of proprioception in patients with meniscus injuries did not influence postural stability. Patients with functional knee instability must therefore be examined for the presence of further injuries because a meniscal lesion alone cannot explain instability symptoms.     

Human Knee Meniscus Regeneration Strategies: a Review on Recent Advances

Purpose of Review

Lack of vascularity in the human knee meniscus often leads to surgical removal (total or partial meniscectomy) in the case of severe meniscal damage. However, complete recovery is in question after such removal as the meniscus plays an important role in knee stability. Thus, meniscus tissue regeneration strategies are of intense research interest in recent years.

Recent Findings

The structural complexity and inhomogeneity of the meniscus have been addressed with processing technologies for precisely controlled three dimensional (3D) complex porous scaffold architectures, the use of biomolecules and nanomaterials.

Summary

The regeneration and replacement of the total meniscus have been studied by the orthopedic and scientific communities via successful pre-clinical trials towards mimicking the biomechanical properties of the human knee meniscus. Researchers have attempted different regeneration strategies which contribute to in vitro regeneration and are capable of repairing meniscal tears to some extent. This review discusses the present state of the art of these meniscus tissue engineering aspects.

     

Biomechanical consequences of a posterior root tear of the lateral meniscus: stabilizing effect of the meniscofemoral ligament

Purpose

The purpose of this study was to evaluate the effects of different types of lateral meniscus root tears in terms of tibiofemoral contact stress.

Methods

Ten porcine knees each underwent five different testing conditions with the menisci intact, a simulated lateral posterior root tear with and without cutting the meniscofemoral ligament and with an artificial tear of the posterior root of the medial meniscus. Biomechanical testing was performed at 30° of flexion with an axial load of 100 N. A pressure sensor (st Sensor Type S2042, Novel, Munich) was used to measure the tibiofemoral contact area and the tibiofemoral contact pressure. Data were analyzed to assess the differences in contact area and tibiofemoral peak contact pressure among the five meniscal conditions.

Results

There was no significant difference in mean contact pressure between the state with the menisci intact and an isolated posterior root tear of the lateral meniscus. In case of a root tear and a tear of the meniscofemoral ligament, the contact area decreased in comparison with the intact state of the menisci. After additional cutting of the meniscofemoral ligament, the tibiofemoral contact pressure was significantly higher in comparison with the intact state and the avulsion injury. In the medial compartment, joint compression forces were significantly increased in comparison with the intact state after cutting the posterior root of the medial meniscus (P < 0.05).

Conclusions

The consequence of a medial meniscus root tear is well known and was verified by this analysis. The results of the present study show that the biomechanical consequences of a lateral meniscus root tear depend on the state of the meniscofemoral ligament. An increase in tibiofemoral contact pressure is only to be expected in combined injuries of the meniscus root and the meniscofemoral ligaments.

Clinical relevance

Posterior lateral meniscus root tear might have a better prognosis in terms of the development of osteoarthritis when the meniscofemoral ligament is intact.     

Clinical results of arthroscopic all-inside lateral meniscal repair using the Meniscal Viper Repair System

Purpose

This study examined the postoperative outcome of the all-inside arthroscopic meniscal suture repair technique using the Meniscal Viper Repair System for lateral meniscus tears.

Methods

Between January 2006 and December 2008, 57 patients (27 males and 30 females) with lateral meniscus tears who underwent meniscal repair using the Meniscal Viper Repair System were evaluated prospectively. Among them, 52 cases were tears involving the posterior third of the lateral meniscus, and most were longitudinal tears or bucket handle tears. The postoperative follow-up period ranged from 12 months to 4 years and 2 months, and averaged 19 months.

Results

Both Lysholm and Japanese Orthopaedic Association Score were significantly improved after surgery. Forty-nine of 57 patients (86.0 %) had no locking or catching, no joint line tenderness, and a negative McMurray test at the last follow-up. Of 32 patients who underwent second-look arthroscopy, 24 achieved complete healing, while four showed partial healing and required repeat repair, and four showed no healing and meniscectomy was conducted. The failure rate in all patients was thus 14.0 % (8 of 57 repairs). No major complications associated with the use of the Meniscal Viper Repair System were observed.

Conclusion

The Meniscal Viper System is a convenient and effective device for all- inside lateral meniscal suture, with high success rate and no major complications.     

Wnt pathway genes in osteoporosis and osteoarthritis: differential expression and genetic association study

Summary

In comparison with hip fractures, increased expression of genes in the Wnt pathway and increased Wnt activity were found in bone samples and osteoblast cultures from patients with osteoarthritis, suggesting the involvement of this pathway in subchondral bone changes. No consistent differences were found in the genetic association study.

Introduction

This study aims to explore the allelic variations and expression of Wnt pathway genes in patients with osteoporosis and osteoarthritis.

Methods

The expression of 86 genes was studied in bone samples and osteoblast primary cultures from patients with hip fractures and hip or knee osteoarthritis. The Wnt-related activity was assessed by measuring AXIN2 and in transfection experiments. Fifty-five SNPs of the LRP5, LRP6, FRZB, and SOST genes were analyzed in 1,128 patients.

Results

Several genes were differentially expressed in bone tissue, with the lowest values usually found in hip fracture and the highest in knee osteoarthritis. Overall, seven genes were consistently upregulated both in tissue samples and in cell cultures from patients with knee osteoarthritis (BCL9, FZD5, DVL2, EP300, FRZB, LRP5, and TCF7L1). The increased expression of AXIN2 and experiments of transient transfection of osteoblasts with the TOP-Flash construct confirmed the activation of Wnt signaling. Three SNPs of the LRP5 gene and one in the LRP6 gene showed marginally significant differences in allelic frequencies across the patient groups, but they did not resist multiple-test adjustment.

Conclusions

Genes in the Wnt pathway are upregulated in the osteoarthritic bone, suggesting their involvement not only in cartilage distortion but also in subchondral bone changes.     

The association between meniscal subluxation and cartilage degeneration

Purpose

The menisci play a critical protective role for the knee joint through shock absorption and load distribution. We hypothesized that cartilage degeneration will be abruptly progressed if meniscal subluxation exceeds a critical point.

Methods

Of 56 cases that showed medial meniscal subluxation without cartilage degeneration of ipsilateral medial femoral condyle (MFC) on initial MRI, from January 2005 to June 2007, meniscal subluxation index (MSI), the ratio of meniscal overhang to meniscal width in mid-coronal image of initial MRI, was measured. After 2 years, 40 cases were evaluated for cartilage degeneration of ipsilateral MFC on follow-up MRI. The relationship between medial MSI on initial MRI and cartilage degeneration of MFC on follow-up MRI was analyzed. Logistic regression analysis was conducted to find a critical point of meniscal subluxation related to cartilage degeneration.

Results

Abrupt progression of cartilage degeneration was observed from which MSI was 0.38. Logistic regression showed that if MSI was at the critical point, which was 0.38 in our study, then the probability of cartilage degeneration to grade 3 or 4 after 2 years was 44 %. If MSI was 0.4, then the probability was 50 %. If MSI was 0.6, then the probability was 99 %.

Conclusions

The results suggest the existence of critical point from which the protective function of the meniscus appears to be significantly altered, and the degree of cartilage degeneration of ipsilateral femoral condyle corresponding to the amount of medial meniscal subluxation may be predictable.     

Evaluation of fibrin-based gene-activated matrices for BMP2/7 plasmid codelivery in a rat nonunion model

Purpose

Treatment of large-segmental bone defects still is a challenge in clinical routine. Application of gene-activated matrices (GAMs) based on fibrin, bone morphogenic protein (BMP) 2/7 plasmids and nonviral transfection reagents (cationic polymers) could be an innovative treatment strategy to overcome this problem. The aim of this study was to determine the therapeutic efficacy of fibrin GAMs with or without additional transfection reagents for BMP2 and 7 plasmid codelivery in a femur nonunion rat model.

Methods

In this experimental study, a critical-sized femoral defect was created in 27 rats. At four weeks after the surgery, animals were separated into four groups and underwent a second operation. Fibrin clots containing BMP2/7 plasmids with and without cationic polymer were implanted into the femoral defect. Fibrin clots containing recombinant human (rh) BMP2 served as positive and clots without supplement as negative controls.

Results

At eight weeks, animals that received GAMs containing the cationic polymer and BMP2/7 plasmids showed decreased bone volume compared with animals treated with GAMs and BMP2/7 only. Application of BMP2/7 plasmids in fibrin GAMs without cationic polymer led to variable results. Animals that received rhBMP2 protein showed increased bone volume, and osseous unions were achieved in two of six animals.

Conclusions

Cationic polymers decrease therapeutic efficiency of fibrin GAM-based BMP2/7 plasmid codelivery in bone regeneration. Nonviral gene transfer of BMP2/7 plasmids needs alternative promoters (e.g. by sonoporation, electroporation) to produce beneficial clinical effects.     

Cell distribution and regenerative activity following meniscus replacement

Purpose

Meniscus replacement is of clinical benefit, but universal efficacy remains elusive. A greater understanding of the biological activity within implanted allografts or synthetic scaffolds may assist the development of improved surgical strategies.

Materials

Biopsies of fresh–frozen allograft (n?=?20), viable allograft (n?=?18) and polyurethane scaffolds (n?=?20) were obtained at second-look arthroscopy. Histological evaluation of tissue morphology and cell density/distribution was performed using haematoxylin–eosin (H&E) staining. Immunohistochemistry was used to detect the presence of CD34 (on progenitor cells and blood vessels) and smooth muscle actin (SMA)-positive structures and aggrecan. Collagen presence was investigated using picrosirius red staining.

Results

Cell density in the deep zone of the meniscus replacement was significantly higher in polyurethane scaffolds versus allograft transplants (p?p?p?p?Conclusions Both viable allograft and a polyurethane meniscal scaffold show enhanced morphological, cell-distribution and regenerative patterns over deep-frozen allograft following surgical implantation. Given the limitations in viable allograft availability, these findings support the continued development of synthetic scaffolds for meniscus replacement surgery.     

The effect of complete radial lateral meniscus posterior root tear on the knee contact mechanics: a finite element analysis

Background

In recent years, with technological advances in arthroscopy and magnetic resonance imaging and improved biomechanical studies of the meniscus, there has been some progress in the diagnosis and treatment of injuries to the roots of the meniscus. However, the biomechanical effect of posterior lateral meniscus root tears on the knee has not yet become clear. The purpose of this study was to determine the effect of a complete radial posterior lateral meniscus root tear on the knee contact mechanics and the function of the posterior meniscofemoral ligament on the knee with tear in the posterior root of lateral meniscus.

Methods

A finite element model of the knee was developed to simulate different cases for intact knee, a complete radial posterior lateral meniscus root tear, a complete radial posterior lateral meniscus root tear with posterior meniscofemoral ligament deficiency, and total meniscectomy of the lateral meniscus. A compressive load of 1000 N was applied in all cases to calculate contact areas, contact pressure, and meniscal displacements.

Results

The complete radial posterior lateral meniscus root tear decreased the contact area and increased the contact pressure on the lateral compartment under compressive load. We also found a decreased contact area and increased contact pressure in the medial compartment, but it was not obvious compared to the lateral compartment. The lateral meniscus was radially displaced by compressive load after a complete radial posterior lateral meniscus root tear, and the displacement took place mainly in the body and posterior horn of lateral meniscus. There were further decrease in contact area and increases in contact pressure and raidial displacement of the lateral meniscus in the case of the complete posterior lateral meniscus root tear in combination with posterior meniscofemoral ligament deficiency.

Conclusions

Complete radial posterior lateral meniscus root tear is not functionally equivalent to total meniscectomy. The posterior root torn lateral meniscus continues to provide some load transmission and distribution functions across the joint. The posterior meniscofemoral ligament prevents excessive radial displacement of the posterior root torn lateral meniscus and assists the torn lateral meniscus in transmitting a certain amount of stress in the lateral compartment.     

Usefulness of knee arthroscopy for diagnosis of knee pain in pediatric patients: comparison with preoperative clinical diagnosis

Purpose

To investigate the knee arthroscopic findings of pediatric patients with knee pain.

Subjects

Ninety-five knees of 94 patients (46 males and 48 females) aged 15 years or younger who underwent knee arthroscopy during a 4-year period from January 2007 were studied. The mean age at surgery was 13.5 (7–15) years. The mean interval from symptom onset to arthroscopic examination was 6.8 months (5 days to 2 years 10 months).

Results

The most common cause of knee pain was sports-related activities (64 knees). Other causes included falling from a moving bicycle (5 knees), while knee pain appeared with no defined reason in 14 knees. The most frequent final diagnosis based on knee arthroscopic findings was anterior cruciate ligament (ACL) injury (35 knees), followed by discoid lateral meniscus (16 knees), lateral meniscal tear (11 knees), and medial plica syndrome (9 knees), while no arthroscopic abnormality was observed in 8 of 95 knees. Among the 95 knees, the diagnosis based on preoperative physical tests and imaging findings was different from the arthroscopic diagnosis in 16 knees, 8 of which were diagnosed preoperatively as medial meniscal tear.

Conclusion

ACL injury and discoid lateral meniscus were the predominant conditions in pediatric patients who underwent knee arthroscopic surgery for knee pain. Knee arthroscopy is useful to provide a definitive diagnosis for knee pain in pediatric patients. Preoperative evaluations had a diagnostic accuracy of only 83.2 % and failed to diagnose conditions such as medial plica syndrome and chondral injury. Therefore, diagnosis before knee arthroscopy has to be interpreted with caution.     

The distribution of vascular endothelial growth factor in human meniscus and a meniscal injury model

Background

The meniscus plays an important role in controlling the complex biomechanics of the knee. Meniscus injury is common in the knee joint. The perimeniscal capillary plexus supplies the outer meniscus, whereas the inner meniscus is composed of avascular tissue. Angiogenesis factors, such as vascular endothelial growth factor (VEGF), have important roles in promoting vascularization of various tissues. VEGF-mediated neovascularization is beneficial to the healing of injured tissues. However, the distribution and angiogenic role of VEGF remains unclear in the meniscus and injured meniscus. We hypothesized that VEGF could affect meniscus cells and modulate the meniscus healing process.

Knee sonography as a diagnostic test for medial meniscal tears in young patients

Purpose

To prospectively evaluate whether age of patient affects diagnostic accuracy of sonography and magnetic resonance imaging (MRI) in the diagnosis of medial meniscal tears.

Methods

We prospectively evaluated 74 consecutive patients (54 males and 20 females), in two different groups [group A (37 patients ≤ 30 years; mean age: 23.5 ± 5 years) and group B (37 patients > 30 years; mean age: 43.5 ± 9.35 years)] with clinical suspicion of medial meniscal tear. After inclusion, patients underwent ultrasonography and then MRI for signs of tearing. The ultrasonographic and MRI findings were compared with arthroscopic findings, which served as a gold standard for accurate detection of meniscal tearing.

Results

The sensitivity, specificity, positive and negative predictive values and accuracy of ultrasonography in detecting medial meniscal tears in group A were 100, 88.9, 96.5, 100, 97.3 % and in group B were 83.3, 71.4, 92.6, 50, 81.1 %, respectively. The sensitivity, specificity, positive and negative predictive values and accuracy of MRI in group A were 100, 88.9, 96.5, 100, 97.3 % and in group B were 96.7, 85.7, 96.7, 85.7, 94.6 %, respectively.

Conclusions

Given the fact that the sensitivity and specificity of the results of knee sonography matched that of MRI in patients who were 30 years old or less, we suggest ultrasonography as an effective initial investigation for tears of medial meniscus in this group of patients. Patients with negative ultrasonographic findings will need no further investigation.

Level of evidence

Diagnostic studies—investigating a diagnostic test, Level II.     

Meniscus suture provides better clinical and biomechanical results at 1-year follow-up than meniscectomy

Background

Surgery of meniscus tear results in limitation of function. The aim of study was functional assessment of knee 1 year after surgery with two techniques in cases of the medial meniscus tear followed by the same supervised rehabilitation.

Materials and methods

A total of 30 patients with good KOSS scores constituted two equal groups after partial meniscectomy or meniscus suture. Measurements of knee extensors and flexors muscles peak torques were performed with angular velocities 60, 180, 240 and 300 s^?1 using Biodex IV system. One-leg-hop and one-leg-rising tests ascertained the function of operated knee. Results of examinations were compared with reference to healthy volunteers. Results of biomechanical and clinical studies were correlated to create complex and objective method evaluating treatment.

Results

Extensors peak torque values at 60 s^?1 angular velocity and H/Q coefficient were decreased after meniscectomy more than meniscus suture in comparison to healthy volunteers (P ≤ 0.001; P ≤ 0.05). Analysis of functional tests revealed that patients after meniscectomy showed difference between operated and non-operated knee (P ≤ 0.01) while patients with meniscus suture differed the least to controls (P ≤ 0.05). Extensors peak torque values at 60 s^?1 angular velocity correlated with results of one-leg-rising test.

Conclusion

Results suggest worse functional effects when meniscectomy is applied which implies modification of the rehabilitative methods in a postoperative period.