David R. Diduch

Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization.

Two cloned cell lines were isolated from cultures of mouse bone-marrow cells. One of the lines, D1, exhibited osteogenic properties and synthesized type-I collagen (alpha 1)2 alpha 2. The second cell line, D2, was not osteogenic and produced a collagen homotrimer (alpha 1)3. Whereas the extracellular matrix of the D1 cell cultures contained striated collagen fibrils, presumably composed of type-I collagen, the homotrimer-producing D2 cells did not demonstrate striated collagen fibrils. Instead, they had thin filaments without detectable striations. Sodium ascorbate stimulated collagen synthesis at the transcriptional level in both the D1 and the D2 cells. The bone-producing characteristics of D1 in vitro included high levels of alkaline phosphatase, increased cyclic adenosine monophosphate on treatment with parathyroid hormone, and expression of osteocalcin mRNA. The D1 cells, unlike the D2 cells, produced a mineralized matrix in vitro. Mineralization in the cultures of the D1 cells occurred in nodules of increased cell density, which also contained the cells with the highest concentrations of collagen mRNA, as shown by in situ hybridization. When the D1 cells were implanted in a diffusion chamber in vivo, a mixture of both osteogenic and adipogenic tissues was formed. This indicates that the D1 cell line is derived from an early marrow stromal precursor that is multipotential.

Chondrocyte transplantation into articular cartilage defects with use of calcium alginate: the fate of the cells.

BACKGROUND The fate of transplanted chondrocytes used to elicit the repair of osteochondral defects is unknown. The objective of this study was to examine the fate and the expression of cartilage-specific genes in chondrocytes when the chondrocyte phenotype was maintained preoperatively by alginate suspension culture, the cells were labeled with enhanced green fluorescent protein, and the chondrocytes in alginate were then implanted into full-thickness osteochondral defects in rabbits. METHODS To determine the effect of alginate on rabbit chondrocytes in vitro, cells were grown in monolayer or in alginate suspension culture, and gene expression for aggrecan, type-I collagen, and type-II collagen was analyzed by reverse transcription-polymerase chain reaction. Cells were genetically labeled with the gene for enhanced green fluorescent protein, and the effect of transfer of the gene for enhanced green fluorescent protein on chondrocyte phenotype was assessed in vitro. Chondrocytes labeled with enhanced green fluorescent protein that were embedded in alginate were implanted into osteochondral defects in rabbit knees, either immediately after creation of the defects or after the cells had been preconditioned in alginate suspension culture for two weeks. The repair tissue within the osteochondral defects was assessed at one to four weeks. Cells labeled with enhanced green fluorescent protein were quantified by confocal microscopy, and the repair tissue was examined histologically with safranin O. RESULTS Gene expression by chondrocytes demonstrated a selective upregulation of cartilage-specific genes in alginate suspension culture. This effect was less pronounced in cells that were transduced with enhanced green fluorescent protein. Chondrocytes transplanted in vivo were detected in the repair tissue for the entire period of observation with diminishing cell density over time. At one week, the cell density of the transplanted chondrocytes was 100% of the initial density; at two and three weeks, the cell density was 70%; and, after four weeks, the cell density had decreased to 15%. Safranin-O staining of histological sections indicated cartilage-specific matrix production in vitro and in vivo. Integration of transplanted cells into the host repair tissue was not observed. The two-week period of preconditioning in alginate suspension culture had no apparent influence on the temporal fate of the cells or the histological appearance of the repair tissue. CONCLUSIONS AND CLINICAL RELEVANCE Alginate promotes expression of cartilage-specific genes and allows delivery of chondrocytes into osteochondral defects. Transgenic chondrocytes labeled with enhanced green fluorescent protein are detectable in the defect, but they do not appear to form repair tissue and they decrease in number with time. In view of the clinical application of cell-based cartilage repair, understanding the fate of transplanted cells becomes increasingly relevant. Transgenic chondrocytes are an effective tool to study the role of transplanted chondrocytes in articular cartilage repair.

Complications Related to Anatomic Reconstruction of the Coracoclavicular Ligaments

Background: Anatomic reconstruction of the coracoclavicular (CC) ligaments has become a popular surgical treatment for high-grade acromioclavicular (AC) dislocations, but little has been reported about complications related to these newer surgical techniques. Purpose: We sought to review the complications related to several new techniques for the anatomic reconstruction of the CC ligaments for the treatment of AC separations. Study Design: Case series; Level of evidence, 4. Methods: We conducted a retrospective review of the operative treatment of AC separation utilizing anatomic reconstruction of the CC ligaments by reviewing the case logs of 3 fellowship-trained orthopaedic surgeons at a single academic sports medicine center for the past 5 years using appropriate current procedural terminology codes. The medical records and postoperative radiographs were assessed for complications. Results: Twenty-seven cases of anatomic reconstruction of the CC ligaments were reviewed. All patients had an autograft or allograft ligament reconstruction utilizing either a coracoid tunnel (10 cases) or a loop around the coracoid base (17 cases). Eight complications (80%) were noted in the coracoid tunnel group including 2 coracoid fractures (20%), 5 patients with some loss of reduction (more than 5-mm increased CC interval displacement on subsequent postoperative radiographs) (50%), and 1 patient with an intraoperative failure of the coracoid button fixation (10%). Six patients developed complications in the coracoid loop group (35%) including 3 clavicle fractures (18% within group, 11% overall), 1 patient with loss of reduction (6%), 1 patient with loss of reduction and an infection (6% within group, 4% overall), and 1 patient with adhesive capsulitis postoperatively (6% within group, 4% overall). Conclusion: Newer techniques for the anatomic reconstruction of the CC ligaments may have steep learning curves associated with complications such as coracoid and clavicle fractures. Loss of reduction continues to be associated with the operative treatment of high-grade AC separations. Further refinement of surgical technique and experience with the operative treatment of AC separation is warranted.

Predictors for Hamstring Graft Diameter in Anterior Cruciate Ligament Reconstruction

Background The ability to accurately predict the diameter of autograft hamstring tendons has implications for graft choice and fixation devices used in anterior cruciate ligament (ACL) reconstruction. Purpose To determine whether simple anthropometric measurements such as height, mass, body mass index (BMI), age, and gender can be used to accurately predict the diameter of hamstring tendons for ACL reconstruction surgery. Study Design Cohort study (prevalence); Level of evidence, 2. Methods The authors conducted medical record reviews and telephone interviews of 106 consecutive patients with ACL reconstruction using quadrupled semitendinosus-gracilis autograft from 2004 to 2006. Data included anthropometric measurements (height, mass, gender, and age at the time of surgery). Hamstring diameter was obtained using cylindrical sizers in 0.5-mm increments and recorded in the patients surgical record. Correlation coefficients (Pearson r) and stepwise, multiple linear regression were used to determine the relationship between the outcome variable (hamstring graft diameter) and the predictor variables (age, gender, height, mass, and BMI). Independent sample t tests were used to compare hamstring graft diameter between genders. Results Hamstring graft diameter was related to height (r = .36, P < .001), mass (r = .25, P = .005), age (r = —.16, P = .05), and gender (r = —.24, P = .006) but was not related to BMI (P > .05). Height was a statistically significant prediction variable (R2 = .13, P < .001). From the current data, a regression equation was calculated that suggested that a patient <147 cm (58 in) tall is likely to have a quadrupled hamstring graft diameter <7 mm in diameter (graft size = 2.4 + 0.03 × height in cm). Women had significantly smaller hamstring graft diameters (7.5 ± 0.7 mm) than did men (7.9 ± 0.9 mm, P = .01). Conclusions Of the parameters studied, height was the best predictor of hamstring tendon diameter, particularly in women.

Deteriorating Outcomes After Meniscal Repair Using the Meniscus Arrow in Knees Undergoing Concurrent Anterior Cruciate Ligament Reconstruction Increased Failure Rate With Long-term Follow-up

Background An increased awareness of the degenerative changes that occur in the knee after meniscectomy has led to efforts to salvage the injured meniscus. Numerous devices have been developed in an effort to provide the dual benefits of a durable meniscal repair and minimal invasiveness. Hypothesis The Meniscus Arrow is comparable to conventional inside-out suture repair in accomplishing long-term healing of meniscal tears. Study Design Case series; Level of evidence, 4. Methods This study is an extended follow-up of an original series of 32 patients withoutcomes analysis. All patients underwent meniscal repair with exclusive use of the arrow. All repairs were performed in the context of a concomitant anterior cruciate ligament reconstruction. Follow-up assessment included physical examination, arthrometry, the International Knee Documentation Committee instrument, and the Knee Disorders Subjective History visual analog scale. Intermediate follow-up at a mean of 2.3 years yielded a success rate of 90.6%. The mean follow-up in the present study has been extended to 6.6 years. Results The extended follow-up analysis revealed a substantial attrition in the success rate of this series of patients undergoing meniscal repair with the arrow. A 90.6% success rate at a mean follow-up of 2.3 years deteriorated to 71.4% at 6.6 years. Conclusion This study provides the longest follow-up in the literature of any of the all-inside meniscal repair implants. The Meniscus Arrow demonstrated long-term meniscal healing rates inferior to those found in the literature for inside-out suture repair techniques.

Systemically administered mesenchymal stromal cells transduced with insulin-like growth factor-I localize to a fracture site and potentiate healing.

Objective To determine to the ability of systemically administered pluripotential mesenchymal stromal cells to localize to a fracture site and whether transduction with a therapeutic gene, insulin-like growth factor-I (D1-IGF-I), could potentiate healing. Design Murine model, basic science study. Setting Laboratory. Specimens Closed, transverse, mid-shaft femur fractures were produced in 108 Balb/c mice after intramedullary stabilization. Interventions A cloned, pluripotential, mesenchymal cell line, termed D1, was stably transfected with either the gene &bgr;-galactosidase (D1-BAG) as a histologic marker or with the gene IGF-I (D1-IGF-I) growth factor. Mice received systemic injections of either D1-BAG cells for in vivo localization or D1-IGF-I for therapeutic intervention. A third group received lactated Ringers solution and served as control. Main Outcome Measurements Sections obtained from the fracture site and contralateral femurs were examined histologically and by deoxyribonucleic acid-polymerase chain reaction (DNA-PCR) to detect the presence of transplanted cells at 2, 4, and 6 weeks after fracture. Matrix mineralization and callus maturation were evaluated by histology. Results At all time points, using histologic staining with X-gal and deoxyribonucleic acid-polymerase chain reaction for marker genes, there was a statistically greater number of transplanted cells (p < 0.001) and significantly higher DNA-PCR for marker genes (p < 0.001) in the fractured femurs than in the nonfractured femurs. Mice receiving D1-IGF-I cells demonstrated a greater percent of mineralized callus than controls at two weeks (p < 0.05). At 4 and 6 weeks, treated mice demonstrated on average greater mineralized matrix and accelerated progression to an osseous callus as compared with the control group. Conclusions Cell-based gene therapy has the potential to deliver higher therapeutic levels of growth factors specifically at the site of cell localization while minimizing wider systemic side effects. This study demonstrates that systemically injected IGF-I transduced, mesenchymal cells are able to return to and repopulate the bone marrow. More importantly, these cells localize preferentially to a fracture site and accelerated fracture healing.

Hamstring Graft Size Prediction A Prospective Clinical Evaluation

Background Recently we retrospectively collected clinical data to predict hamstring graft diameter. Prospective data collection will improve and further define prediction of hamstring graft size. Hypothesis Clinical anthropometric data can be used to predict hamstring graft size. Study Design Cohort study (prevalence); Level of evidence, 1. Methods Fifty consecutive patients with anterior cruciate ligament deficiency scheduled for reconstruction using hamstring autograft were prospectively evaluated. Preoperatively we recorded height, weight, body mass index, age, gender, leg length, thigh length, shank length, bilateral thigh circumference, and Tegner score. Intraoperative measurements of both the gracilis and semitendinosus tendons were made, including absolute length before fashioning the graft and final diameter of the quadrupled graft using sizing tubes calibrated to 0.5 mm. Bivariate correlation coefficients (Pearson r) were calculated to identify relationships among clinical data and intraoperatively measured hamstring graft length and diameter. Results Strongest correlations for graft lengths were height and leg length measurements. Shorter persons with shorter leg, thigh, and shank lengths tended to have shorter gracilis and semitendinosus grafts. Likewise, the strongest correlations for graft diameter were weight and thigh circumference. Self-reported activity level and age did not correlate. Gender comparison revealed that women who were shorter, lighter, and had smaller body mass indices were more likely to have smaller graft diameters and shorter graft lengths. Conclusion Patients weighing less than 50 kg, less than 140 cm in height, with less than 37 cm thigh circumference, and with body mass index less than 18 should be considered at high risk for having a quadrupled hamstring graft diameter less than 7 mm. When separated by gender, small graft diameters are most likely in older, short, female subjects with small thigh circumferences or young, skinny, male subjects with small thigh circumferences and low body mass index. Common clinical measurements can be used for preoperative identification of patients at risk for insufficient graft tissue and would be useful for patient counseling and alternative graft source planning.

Multiple-Ligament Knee Injuries: A Systematic Review of the Timing of Operative Intervention and Postoperative Rehabilitation

BACKGROUND Traumatic knee dislocations that result in multiple-ligament knee injuries are unusual and are poorly studied. We are not aware of any prospective data regarding their treatment. Both the optimum timing of surgery for repair or reconstruction and the aggressiveness of rehabilitation are debated. The purpose of this systematic review was to compare the outcomes of early, delayed, and staged procedures as well as the subsequent rehabilitation protocols. METHODS We surveyed the literature and retrieved twenty-four retrospective studies, involving 396 knees, dealing with the surgical treatment of the most severe multiple-ligament knee injuries (those involving both cruciate ligaments and either or both collateral ligaments). Data were extracted, and surgical timing was categorized as acute, chronic, or staged. Early postoperative mobility and immobilization were also compared. RESULTS We found that acute treatment was associated with residual anterior knee instability when compared with chronic treatment (odds ratio, 2.58; 95% confidence interval, 1.2 to 5.8; p = 0.018). Significantly more patients who were managed acutely were found to have more flexion deficits when compared with those who were managed chronically (odds ratio, 5.18; 95% confidence interval, 1.5 to 17.5; p = 0.004). Staged treatments yielded the highest percentage of excellent and good subjective outcomes (79%; 95% confidence interval, 62.2% to 89.3%). Additional treatment for joint stiffness was significantly more likely in association with acute treatment (17%; 95% confidence interval, 13.0% to 22.4%; p < 0.001) and staged treatment (15%; 95% confidence interval, 7.6% to 28.2%; p = 0.001) when each was compared with chronic treatment (0% [zero of seventy-one]; 95% confidence interval, 0.0% to 5.1%). Early mobility was not associated with increased joint instability in acutely managed patients. Early mobility yielded fewer range-of-motion deficits but did not reduce the rate of follow-up manipulation or arthrolysis. CONCLUSIONS This review of the available literature suggests that delayed reconstructions of severe multiple-ligament knee injuries could potentially yield equivalent outcomes in terms of stability when compared with acute surgery. However, in the acutely managed patient, early mobility is associated with better outcomes in comparison with immobilization. Acute surgery is highly associated with range-of-motion deficits. Staged procedures may produce better subjective outcomes and a lower number of range-of-motion deficits but are still likely to require additional treatment for joint stiffness. More aggressive rehabilitation may prevent this from occurring in multiple-ligament knee injuries that are treated acutely.

The use of absorbable co-polymer pads with alginate and cells for articular cartilage repair in rabbits.

PURPOSE To determine the effect of polyglycolic acid (PGA)-polylactic acid (PLA) co-polymer pads with calcium alginate on chondrogenic gene expression for chondrocytes cultured in vitro. We also evaluated the ability of these absorbable pads with alginate to deliver chondrocytes and influence osteochondral defect repair in vivo in immature rabbit knees. METHODS Rabbit rib chondrocytes were suspended in calcium alginate and co-polymer pads composed of either 47.5/52.5 PGA-PLA or 90/10 PGA-PLA at two different cell concentrations and cultured in vitro for 1, 3, and 5 days. Analysis was performed using RT-PCR for chondrogenic gene expression of aggrecan, type II collagen, and type I collagen. Cells labeled with a traceable green fluorescent protein (GFP) marker in vitro were suspended within the pads to analyze for dispersion and attachment to the pad. An in vivo study was performed in which full-thickness (3x4mm(2)) osteochondral defects were made in 60 rabbit knees. The study comprised four treatment groups based on the type of implant into the defect (empty, alginate alone, or either type of co-polymer pad) and harvested at either 6 or 12 weeks. Two independent blinded observers analyzed and scored the defects grossly and histologically. RESULTS In vitro analysis of the chondrocytes after 1, 3, and 5 days in culture showed no statistical differences between the types of PGA/PLA co-polymer pad with regard to expression of aggrecan, type II collagen, or type I collagen. However, although statistically insignificant, the expression of aggrecan and type II collagen was found to be greater than that for type I collagen in both types of pads, confirming the chondrogenic effect of suspension culture for this system. The addition of alginate to polymer pads allowed costal chondrocytes to be implanted in vivo, as evidenced by the attachment of the cells to the fibers and the uniform dispersion of the GFP-labeled cells through the pad as seen on fluorescent microscopy. Histologic results showed improved scores for the 47.5/52.5 PGA-PLA group (21.3) and the 90/10 PGA-PLA group (18.3) when compared to empty (15.3) or alginate alone (15.1) defects at 12 weeks. CONCLUSION The addition of calcium alginate to the co-polymer pads offers a new approach to deliver cells to an osteochondral defect and may enhance cartilage regeneration. Future application of this model may allow for an arthroscopic delivery system to assist the healing of cartilage defects.

All-Inside Meniscal Repair Using a New Flexible, Tensionable Device

Background A new generation of flexible all-inside meniscal repair devices is available, but clinical studies with these devices are lacking. Hypothesis The RapidLoc has an intermediate-term meniscal healing rate that is equivalent to literature reports of inside-out suture technique in patients undergoing concurrent anterior cruciate ligament reconstruction. Study Design Case series; Level of evidence, 4. Methods Retrospective analysis was performed for 75 meniscal tears in 66 consecutive patients undergoing anterior cruciate ligament reconstruction who underwent meniscal repair with the RapidLoc. Patients with at least 2 years’ follow-up were evaluated for symptoms suggestive of a meniscal tear and were assessed with the International Knee Documentation Committee form and the Knee Disorders Subjective History visual analog scale. Subjects were asked to return for a clinical examination to include evaluation for an effusion and joint line tenderness as well as McMurray test and KT-1000 arthrometry readings. Patients with symptoms consistent with meniscal repair failure underwent magnetic resonance arthrography and repeat arthroscopy. Results Twenty patients with 21 meniscal tears were excluded, resulting in 54 meniscal tears in 46 patients. At a mean follow-up of 34.8 months, the clinical success rate for meniscal repair was 90.7% (49/54), with 5 failures requiring meniscectomy. Univariate analysis revealed predictive variables for failure: bucket-handle configuration, multiplanar tears, tear length greater than 2 cm, and chronicity longer than 3 months. Nonpredictive variables included compartment, zone, ligament graft choice, gender, age, follow-up, and visual analog scale score. Analysis of healed patients revealed a negative correlation between chronicity of tear and International Knee Documentation Committee score. Conclusions The RapidLoc has an acceptable intermediate-term clinical healing rate in patients undergoing concurrent anterior cruciate ligament reconstruction. Predictive variables for failure should be considered during operative decision making.