Direct incorporation of mesenchymal stem cells into a Nanofiber scaffold - in vitro and in vivo analysis.

Bony defects are a common problem in musculoskeletal surgery. Replacement with autologous bone grafts is limited by availability of transplant material. Sterilized cancellous bone, while being osteoconductive, has limited osteoinductivity. Nanofiber scaffolds are currently used for several purposes due to their capability of imitating the extracellular matrix. Furthermore, they allow modification to provide functional properties. Previously we showed that electrospun nanofiber scaffolds can be used for bone tissue regeneration. While aiming to use the osteoinductive capacities of collagen type-I nanofibers we saw reduced scaffold pore sizes that limited cellular migration and thus colonization of the scaffolds. Aim of the present study was the incorporation of mesenchymal stem cells into the electrospinning process of a nanofiber scaffold to produce cell-seeded nanofiber scaffolds for bone replacement. After construction of a suitable spinning apparatus for simultaneous electrospinning and spraying with independently controllable spinning and spraying devices and extensive optimization of the spinning process, in vitro and in vivo evaluation of the resulting scaffolds was conducted. Stem cells isolated from rat femora were incorporated into PLLA (poly-l-lactide acid) and PLLA-collagen type-I nanofiber scaffolds (PLLA Col I Blend) via simultaneous electrospinning and -spraying. Metabolic activity, proliferation and osteoblastic differentiation were assessed in vitro. For in vivo evaluation scaffolds were implanted into critical size defects of the rat scull. After 4 weeks, animals were sacrificed and bone healing was analyzed using CT-scans, histological, immunhistochemical and fluorescence evaluation. Successful integration of mesenchymal stem cells into the scaffolds was achieved by iteration of spinning and spraying conditions regarding polymer solvent, spinning distance, the use of a liquid counter-electrode, electrode voltage and spinning duration. In vivo formation of bone tissue was achieved. Using a PLLA scaffold, comparable results for the cell-free and cell-seeded scaffolds were found, while the cell-seeded PLLA-collagen scaffolds showed significantly better bone formation when compared to the cell-free PLLA-collagen scaffolds. These results provide support for the future use of cell-seeded nanofiber scaffolds for large bony defects.

Rasterstereographic Analysis of Lateral Shift in Patients with Lumbar Disc Herniation: A Case Control Study.

OBJECTIVE: Detection of a lateral shift (LS) in patients with diagnosed disc herniation compared to healthy controls. SUMMARY OF BACKGROUND DATA: A specific lateral shift (LS) pattern is observed in patients with disc herniation and low back pain, as shown in earlier studies. METHODS: Rasterstereography (RS) was used to investigate the LS. Thirty-nine patients with lumbar disc herniation diagnosed by radiological assessment and low back pain and/or leg pain (mean age 48.2 years, mean BMI 28.5, 28 males and 11 females) and 36 healthy controls (mean age 47.4 years, mean BMI 25.7, 25 males and 11 females) were analysed. LS, pelvic tilt, pelvic inclination, lordotic angle, and trunk torsion were assessed. RESULTS: The patient group showed a nonsignificant increase in LS, that is, 5.6 mm compared to the healthy controls with 5.0 mm (p = 0.693). However, significant differences were found between groups regarding pelvic tilt in degrees (patients 5.9°, healthy controls 2.0°; p = 0.016), trunk torsion (patients 7.5°, controls 4.5°; p = 0.017), and lordotic angle (patients 27.5°, healthy controls 32.7°; p = 0.022). The correlation between pain intensity and the FFbH-R amounted 0.804 (p = < 0.01), and that between pain intensity and the pain disability index was 0.785 (p < 0.01). DISCUSSION: Although some studies have illustrated LS with disc herniation and low back pain, the present findings demonstrate no significant increase in LS in the patient group compared to healthy controls. CONCLUSION: The patients with lumbar disc herniation did not demonstrate an increased LS compared to healthy controls. Other parameters like pelvic tilt and inclination seemed to be more suitable to identify changes in posture measured by RS in patients with low back pain or disc herniation.

Expression of MMP-9 decreases metastatic potential of Chondrosarcoma: an immunohistochemical study.

BACKGROUND: Chondrosarcoma is the second most common primary malignant bone tumor. Because of their heterogeneity, with differences in invasive and metastatic behavior, it is important to identify biological markers that will allow for a more accurate estimation of prognosis in patients with these tumors. Matrix metalloproteinases (MMP) play a crucial role in tumor progression, invasion and metastasis. The mechanism of tumor progression dependent of MMPs is complex and influences malignant transformation, angiogenesis and tumor growth at the primary and metastatic sites. The purpose of this study was to investigate immunohistochemicaly the influence of MMP-1, MMP-3, MMP-9 and MMP-13 expression on prognostic parameter in chondrosarcoma. METHODS: We investigated tissue samples of 28 patients with chondrosarcoma. Immunohistochemical staining to evaluate the expression of MMP-1, MMP-3, MMP-9 and MMP-13 was performed. Subsequently, the expression level was correlated with metastatic potential, histological grading and overall survival in patients with this neoplasm. RESULTS: In consideration of semi quantitative scoring 64% of chondrosarcoma were scored as positive for MMP-1, 46% for MMP-3, 61% for MMP-9. The specimens had shown no expression of MMP-13. High expression of MMP-9 was associated with better histological differentiation, decreased metastatic potential and favourable overall survival. No correlation was found for expression of MMP-1, MMP-3 or MMP-13. CONCLUSIONS: MMP-1, MMP-3 and MMP-9 are expressed in chondrosarcoma. Our findings suggest that the expression of MMP-9 is associated with clinical outcome parameters in chondrosarcoma.

Influence of a Training Session on Postural Stability and Foot Loading Patterns in Soccer Players.

Sport specific movements coming along with characteristic plantar pressure distribution and a fatigue of muscles result in an increasing postural sway and therefore lead to a decrease in balance control. Although single soccer specific movements were expatiated with respect to these parameters, no information is available for a complete training session. The objective of the present observational study was to analyze the direct influence of soccer training on postural stability and gait patterns and whether or not these outcomes were altered by age. One hundred and eighteen experienced soccer players participated in the study and were divided into two groups. Group 1 contained 64 soccer players (age 13.31±0.66 years) and Group 2 contains 54 ones (age 16.74±0.73 years). Postural stability, static plantar pressure distribution and dynamic foot loading patterns were measured. Our results showed that the soccer training session, as well as the age, has relevant influence on postural stability, while the age only (excluding the training) has an influence on static plantar pressure distribution. The parameters of dynamic assessment seem therefore to be affected by age, training and a combination of both. Training and young age correlate with a decreased postural stability; they lead to a significant increase of peak pressure in the previously most loaded areas, and, after reaching a certain age and magnitude of absolute values, to a change in terminal stance and preswing phase of the roll-over. Moreover, younger players show an inhomogenous static plantar pressure distribution which might be the result of the decreased postural control in the young age.

Systemic antibiotic therapy does not significantly improve outcome in a rat model of implant-associated osteomyelitis induced by Methicillin susceptible Staphylococcus aureus.

INTRODUCTION: Treatment of implant-associated osteomyelitis regularly involves the use of systemic antibiotics in addition to surgical intervention. However, it remains unclear if perioperative systemic application of bactericide substances can improve overall outcome in models of severe intramedullary infection. The present study investigated the use of systemic gentamicin in addition to a controlled local release from a highly lipophilic gentamicinpalmitate compound while the previous study showed efficacy of sole antibiotic implant-coating. METHODS: Forty male Sprague-Dawley rats were divided into two groups receiving an intramedullary femoral injection of 10(2) CFU of a common methicillin susceptible Staphylococcus aureus strain (MSSA Rosenbach). Group I received an uncoated implant whereas group II received a coated implant. All animals received a single shot intraperitoneal application of gentamicinsulfate directly after wound closure while the historical control group III (n = 20) had no antibiotic treatment at all. Animals were observed for 28 and 42 days. Serum haptoglobin and relative weight gain were assessed as well as roll over cultures of explanted femur nails and histological scores of periprosthetic infection in dissected femora. RESULTS: Systemic application of gentamicin combined with antibiotic-coated implant did not further reduce bacterial growth significantly compared with systemic or local antibiotic application alone. Combined local and systemic therapy reduced serum haptoglobin significantly after day 7, 28 and 42 whereas systemic application alone did not compare to controls. CONCLUSIONS: Systemic perioperative and implant-associated application of antibiotics were both comparably effective to treat implant-associated infections whereas the combined antibiotic therapy further reduced systemic signs of infection time dependent.

Role of Electrochemically Activated Solution in Asepsis in Osteoblasts and Chondrocytes in vitro.

PURPOSE: Implant infections are severe complications in orthopedic surgery. Treatment using antibiotics is often unsuccessful without removing the implant due to biofilm formation. In this context, antiseptics may be a potential option. These solutions have a wide antimicrobial spectrum but often cause damage to the local cell populations. In these cases, electrochemically activated solutions (ECAS) may be an alternative. METHODS: Staphylococcus aureus was cultured overnight on polyethylene platelets under different growing conditions (standard-/-different biofilm inducing conditions). The samples were treated with an ECAS (0%-30%) and analyzed with regard to their antimicrobial efficacy and their effect on biofilm structures by determining the living bacterial count and scanning electron microscopy. Osteoblast and chondrocyte cell lines (MG-63, and CAL-78, respectively) were cultured to confluence and treated with an ECAS. Total cell count, metabolic activity using FDA staining, and proliferation using Ki67 staining were analyzed. RESULTS: ECAS showed antimicrobial activity in concentrations <1%. In cases of biofilm formation, bacterial toxicity could be detected at concentrations from 3.75 -7.5%. At these same concentrations, a visible change in biofilm structure was observed. Regarding biocompatibility rates, up to 7.5% (of osteoblasts) and 3.75% (of chondrocytes) had little influence on metabolic activity and proliferation. CONCLUSION: ECAS provide good antimicrobial efficacy while maintaining high biocompatibility. These results suggest that these solutions may provide promising alternatives in the treatment of implant-associated infections.

Juvenile osteochondritis dissecans of the talus: predictors of conservative treatment failure.

BACKGROUND: The ideal treatment for juvenile osteochondritis dissecans of the talus (ODT) is still unclear. To determine predictors of failure of conservative treatment, children admitted for ODT were retrospectively analyzed. METHODS: Patient files were analyzed to search for children treated for an ODT between 2000 and 2011. X-rays and MRI at baseline were evaluated for grading of lesions and the patient history was obtained. Final follow-up evaluation was performed via questionnaire and complementary telephone interview. Outcome was measured using the AOFAS and the Olerud/Molander scores. Conservative treatment consisted of out of sports and modification of activity under full weight-bearing. In case of persisting pain, full load removal on crutches was initiated. For further analysis, two groups were formed: (1) successful conservative treatment; (2) converted to surgical therapy. A logistic regression was used to determine potential predictors of conservative treatment failure. RESULTS: Seventy-seven lesions in 67 children with a mean age of 11.4 years (range 4-15 years) at the time of diagnosis were identified. Every patient received conservative treatment as a first-line treatment after diagnosis of ODT except for one single patient with a grade IV lesion at time of diagnosis who received operative treatment directly after diagnosis. Sixty-one percent of the lesions failed conservative treatment. A higher age as well as a grade III lesion at time of diagnosis was predictive for failure of the conservative treatment (p = 0.03 and p = 0.02, respectively). Regarding the functional outcome, a higher grade lesion in general was predictive for an inferior outcome as measured by clinical score. CONCLUSION: Grade III ODT especially in older children leads significantly more often to treatment failure when treated non-surgically. No other predictors for treatment failure could be identified. LEVEL OF EVIDENCE: Level III (retrospective comparative study).

Bleeding in TKA: posterior stabilized vs. cruciate retaining.

INTRODUCTION: Posterior-stabilized (PS) and cruciate-retaining (CR) total knee arthroplasties (TKA) are both successfully used for treatment of end-stage osteoarthritis. The choice of constraint depends on knee deformity and stability as well as most importantly surgeon preference. The aim of this study was to compare the amount of blood loss and required transfusions following TKA with the two different designs. MATERIALS AND METHODS: In a retrospective approach, 473 patients undergoing TKA were included (240 CR and 233 PS from a single manufacturer). Demographics at base line were comparable between both groups. Blood loss [red blood cell (RBC) loss] was calculated after documentation of pre- and postoperative hematocrit levels at discharge. Transfusion requirements were recorded. Statistical analysis was done using Mann-Whitney U test. RESULTS: The calculated blood loss (RBC loss) at discharge was 548 ± 216 ml in the PS group compared with 502 ± 186 ml in the CR group (p = 0.032). There were no differences in the transfusion requirements between both groups (PS 0.41 vs. CR 0.37, p = 0.39). DISCUSSION: The blood loss was significantly higher in the PS group. This may be due to the box preparation that exposes more cancellous femoral bone, which may add to postoperative bleeding. The differences remain, however, small, as they did not lead to a significantly higher transfusion rate with PS TKA.

Coating with a novel gentamicinpalmitate formulation prevents implant-associated osteomyelitis induced by methicillin-susceptible Staphylococcus aureus in a rat model.

PURPOSE: Implant-associated osteomyelitis still represents a demanding challenge due to unfavourable biological conditions, bacterial properties and incremental resistance to antibiotic treatment. Therefore different bactericide or bacteriostatic implant coatings have been developed recently to control local intramedullary infections. Controlled local release of gentamicin base from a highly lipophilic gentamicin palmitate compound achieves extended intramedullary retention times and thus may improve its bactericide effect. METHODS: Forty male Sprague-Dawley rats were divided into two groups receiving an intramedullary femoral injection of 10(2) colony-forming units (CFU) of a common methicillin susceptible Staphylococcus aureus strain (MSSA Rosenbach) and either an uncoated femur nail (Group I) or a nail coated with gentamicin palmitate (Group II). Animals were observed for 28 and 42 days. Serum haptoglobin and relative weight gain were assessed as well as rollover cultures of explanted femur nails and histological scores of periprosthetic infection in dissected femurs. RESULTS: Implants coated with gentamicin palmitate significantly reduced periprosthetic bacterial growth as well as signs of systemic inflammation compared with uncoated implants. CONCLUSIONS: Gentamicin palmitate appears to be a viable coating for the prevention of implant-associated infections. These findings will have to be confirmed in larger animal models as well as in clinical trials.

Expression of hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinases 1, 3, and 9 in hypertrophied ligamentum flavum.

STUDY DESIGN: Immunohistological study. OBJECTIVE: To elucidate the role of matrix metalloproteinases (MMPs), hypoxia-inducible factor-1α (HIF), and vascular endothelial growth factor (VEGF) in the hypertrophied ligamentum flavum (LF) obtained from patients with lumbar spinal stenosis (LSS). SUMMARY OF BACKGROUND DATA: The most common spinal disorder in the elderly is LSS, which results in part from LF hypertrophy. Although prior histologic and immunochemical studies have been performed in this area, the pathophysiology of loss of elasticity and hypertrophy is not completely understood. METHODS: LF samples of 38 patients with LSS were harvested during spinal decompression. Twelve LF samples obtained from patients with disk herniation and no visible degeneration on preoperative magnetic resonance imaging were obtained as controls. Samples were dehydrated and paraffin embedded. For immunohistochemical determination of VEGF, HIF, and MMPs 1, 3, and 9 expression, slices were stained with VEGF, HIF, and MMP antibody dilution. Neovessel density and number of elastic fibers were counted after Masson-Goldner staining. LF hypertrophy and cross-sectional area (CSA) were measured on T1-weighted magnetic resonance imaging. RESULTS: MMPs 1, 3, 9 and VEGF expression were significantly increased in the hypertrophy group (P<0.05). HIF expression was negative in both groups. Vessel density was increased in the hypertrophy group, although this was not statistically significant. The number of elastic fibres was significantly higher in the control group. In the hypertrophy group, LF thickness was significantly increased, whereas CSA was significantly decreased. There was a statistical correlation between LF thickness, CSA, MMP, and VEGF expression in the hypertrophy group (P<0.05). CONCLUSIONS: LF hypertrophy is accompanied by increased MMPs 1, 3, 9 and VEGF expression. Neovessel density is increased in hypertrophied LF. HIF is not expressed in hypertrophied LF.

Correlation of pull-out strength of cement-augmented pedicle screws with CT-volumetric measurement of cement.

BACKGROUND: Cement augmentation of pedicle screws increases fixation strength in an osteoporotic spine. This study was designed to determine the cement distribution and the correlation between the pull-out strength of the augmented screw and the cement volume within polyurethane (PU) foam. METHODS: Twenty-eight cannulated pedicle screws (6×45 mm) (Peter Brehm, Erlangen, Germany) with four holes at the distal end of the screw were augmented with the acrylic Stabilit ER Bone Cement Vertebral Augmentation System (DFine Inc., San Jose, CA, USA) and implanted into open-cell rigid PU foam (Pacific Research Laboratories, Vashon Island, WA, USA) with a density of 0.12 g/cm3, resembling severe osteoporosis. Volumetric measurement of the cement with consideration of the distribution around the screws was done with multislice computed tomography scan (Somatom Definition, Siemens, Erlangen, Germany). Pull-out strength was tested with a servohydraulic system (MTS System Corporation, Eden Prairie, MN, USA), and nonaugmented screws served as control. Pearson's correlation coefficient with significance level α=0.05 and one-way analysis of variance test were used. RESULTS: We found a high (r=0.88) and significant (p<0.01) correlation between the cement volume and the pull-out strength, which increased by more than 5-fold with a volume of 3 ml. The correlation appeared linear at least up to 4 ml cement volume and failure always occurred at the cement-bone interface. The cement distribution was symmetric and circular around the most proximal hole, with a distance of 14 mm from the tip, and nearly 90% of the cement was found 6 mm distal and cranial to it. The 95% confidence interval for the relative amount of cement was 37%-41% within 2 mm of the most proximal hole. CONCLUSION: Compared with the control, a cement volume between 2.0 and 3.0 ml increased the pull-out strength significantly and is relevant for clinical purposes, whereas a volume of 0.5 ml did not. A cement volume beyond 3.0 ml should further increase the pull-out strength because the correlation was linear at least up to 4.0 ml, but the possibility of in vivo cement leakage with increasing volume has to be considered. Pressure-controlled cement application might be a tool to avoid this complication. The cement almost completely penetrated the most proximal perforation.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo.

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.

Cell-free collagen type I matrix for repair of cartilage defects-clinical and magnetic resonance imaging results.

PURPOSE: Several well-described techniques are available for the treatment of chondral and osteochondral defects. The aim of the study was to assess the efficacy of a single-stage procedure incorporating a new cell-free collagen type I gel for the treatment of small chondral and osteochondral defects in the knee evaluated at 2-year follow-up. METHODS: Fifteen patients were treated with a cell-free collagen type I gel matrix of 11 mm diameter. The grafts were implanted in the debrided cartilage defect and fixed by press-fit only. The clinical outcome was assessed preoperatively and at 6 weeks, and 6, 12 and 24 months after surgery using the International Knee Documentation Committee (IKDC) score, Tegner activity scale and visual analogue scale (VAS). Graft attachment rate was assessed 6 weeks postoperatively using magnetic resonance imaging (MRI). Cartilage regeneration was evaluated using the Magnetic Observation of Cartilage Repair Tissue (MOCART) score at 6, 12 and 24 months after implantation. Clinical results were correlated with MRI findings. RESULTS: Six male and nine female patients were included in this study, with a mean age of 26 (range: 19-40). No complications were reported. The mean VAS values after 6 weeks and the mean IKDC patient values after 6 months were significantly improved from the preoperative values (P = 0.005 and P = 0.009, respectively). This improvement remained up to the latest follow-up. There were no significant differences between the median preoperative and postoperative Tegner values (n.s.). Significant improvement of the mean MOCART score was observed after 12 months and remained by 24 months (P < 0.001). MR images showed that in 14 of the 15 patients, the graft was completely attached by 6 weeks postoperatively. At 24 months after implantation, MRI demonstrated complete filling in all cases with a mainly smooth surface, complete integration of the border zone, homogenous structure of the repaired tissue and nearly normal signal intensity. No correlation between any variables of the MOCART score and the clinical scores was observed. CONCLUSIONS: The present study reveals that the new method produces both good clinical and magnetic resonance imaging results. Use of press-fit only implanted grafts of a smaller diameter leads to a high attachment rate at 24-month follow-up. LEVEL OF EVIDENCE: IV.

The safety and short-term efficacy of a novel polyurethane meniscal scaffold for the treatment of segmental medial meniscus deficiency.

PURPOSE: Meniscal loss is associated with an increased risk of developing osteoarthritis. Tissue engineering solutions, which include the development of novel material scaffolds, are being utilised to aid the regeneration of meniscal tissue. The purpose of this study was to monitor the safety profile and the short-term efficacy of a novel polyurethane meniscal scaffold in the treatment of patients with painful knees following partial medial meniscectomy. METHODS: Ten consecutive patients with segmental tissue loss from the medial meniscus were treated with the arthroscopic implantation of an Actifit(®) (Orteq Sports Medicine) polyurethane meniscal scaffold. Patients were followed up at 2 and 6 weeks, then again at 6 and 12 months. Primary outcome measures included reporting of complications and patient-reported outcome scores (KOOS, KSS, UCLA Activity scale, VAS pain). Secondary outcome was MRI assessment at 6 and 12 months looking at scaffold morphology, integration and associated joint injury/inflammation. RESULTS: Eight male and 2 female patients were included in the study with a mean age of 29 (range 18-45). No complications were reported. All patients were happy with their surgery. At 6 months, a statistically significant improvement (P < 0.05) in all PROMS except the UCLA activity scale and VAS pain scale were noted. The improvement remained at 12 months. MRI analysis revealed the presence of scaffolds at 6 months, with evidence of some tissue integration in many improvements in scaffold morphology and ICRS classification of cartilage in the medial compartment were noted at 12 months. No synovitis was noted in the joint or adverse reactions in the other compartments. CONCLUSION: The new Actifit(®) polyurethane scaffold has been shown to be a safe, effective implant, for the treatment of patients with pain as a result of segmental medial meniscus loss at 1 year. LEVEL OF EVIDENCE: IV.

Fibrin glue does not improve the fixation of press-fitted cell-free collagen gel plugs in an ex vivo cartilage repair model.

PURPOSE: Adequate graft fixation over a certain time period is necessary for successful cartilage repair and permanent integration of the graft into the surrounding tissue. The aim of the present study was to test the primary stability of a new cell-free collagen gel plug (CaReS(®)-1S) with two different graft fixation techniques over a simulated early postoperative period. METHODS: Isolated chondral lesions (11 mm diameter by 6 mm deep) down to the subchondral bone plate were created on the medial femoral condyle in 40 porcine knee specimens. The collagen scaffolds were fixed in 20 knees each by press-fit only or by press-fit + fibrin glue. Each knee was then put through 2,000 cycles in an ex vivo continuous passive motion model. Before and after the 2,000 motions, standardized digital pictures of the grafts were taken. The area of worn surface as a percentage of the total collagen plug surface was evaluated using image analysis software. RESULTS: No total delamination of the scaffolds to leave an empty defect site was recorded in any of the knees. The two fixation techniques showed no significant difference in worn surface area after 2,000 cycles (P = n.s.). CONCLUSIONS: This study reveals that both the press-fit only and the press-fit + fibrin glue technique provide similar, adequate, stability of a type I collagen plug in the described porcine model. In the clinical setting, this fact may be particularly important for implantation of arthroscopic grafts.

Increased expression of CD44 in hypertrophied ligamentum flavum and relevance of splice variants CD44v5 and CD44v6.

BACKGROUND: The most common spinal disorder in the elderly is lumbar spinal stenosis (LSS), which results in part from ligamentum flavum (LF) hypertrophy. Although prior histologic and immunochemical studies have been performed in this area, the pathophysiology of loss of elasticity and hypertrophy is not completely understood. The purpose of this immunohistological study is to elucidate the role of CD44 and its splice variants CD44v5 and CD44v6 in the hypertrophied LF obtained from patients with lumbar spinal stenosis (LSS). MATERIALS AND METHODS: LF samples of 38 patients with LSS were harvested during spinal decompression. Twelve LF samples obtained from patients with disc herniation and no visible degeneration on preoperative MRI were obtained as controls. Samples were dehydrated and embedded in paraffin. For immunohistochemical determination, slices were stained with antibodies against CD44, Cd44v4, and CD44v6 stained with DAB. LF hypertrophy and cross-sectional area (CSA) were measured with T1-weighted MRI. RESULTS: CD44 and CD44v5 expression were significantly increased in the hypertrophy group (p < 0.05). CD44v6 expression was not significantly increased. The number of elastic fibers was significantly higher in the hypertrophy group. In the hypertrophy group, LF thickness was significantly increased while CSA was significantly decreased. There was a statistical correlation between LF thickness, CSA, CD44, and CD44v5 expression in the hypertrophy group (p < 0.05). CONCLUSIONS: LF hypertrophy is accompanied by increased CD44 and CD44v5 expression. CD44v6 expression is not enhanced in LF hypertrophy.

Cooperative effects in differentiation and proliferation between PDGF-BB and matrix derived synthetic peptides in human osteoblasts.

BACKGROUND: Enhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF) BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells. METHODS: Osteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin. RESULTS: Experimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05). The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p < 0.05). CONCLUSIONS: Our findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p < 0.05). We suggest the combination of synthetic peptides and PDGF as a potential clinical approach for accelerating bone healing or coating osteosynthesis materials.

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects.

INTRODUCTION: Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). MATERIALS AND METHODS: The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). RESULTS: PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. CONCLUSION: Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.

Initial stability of tibial components in primary knee arthroplasty. A cadaver study comparing cemented and cementless fixation techniques.

The purpose of this roentgen stereophotogrammetric analysis (RSA) study was to evaluate the initial stability of cemented and cementless tibial components in vitro. Twenty tibia specimens were matched into two groups. In the first group, the tibial trays were cemented superficially and in the second group cementless fixation with stem and screws was performed. An axial load of 2000 N for 1000 and 10,000 cycles was applied onto the specimens and RSA was performed. The experimental results after 1000 cycles showed a higher migration with significant differences for the parameters maximum lift off (p = 0.011) and maximum total point motion (p = 0.002) in the cementless group. After 10,000 cycles, the migration in the cementless group increased significantly for maximum lift off (p = 0.043), maximum subsidence (p = 0.045) and maximum total point motion (p = 0.013). The higher migration rates in the cementless group demonstrate a lower initial mechanical stability of cementless tibial components which can cause an early component loosening.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on growth and osteogenic differentiation of human mesenchymal stem cells.

Mesenchymal stem cell differentiation of osteoblasts is triggered by a series of signaling processes including integrin and bone morphogenetic protein (BMP), which therefore act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in an artificial poly-(L)-lactide acid (PLLA) based nanofiber scaffold. Matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were seeded with human mesenchymal stem cells (hMSC) and cultivated over a period of 22 days, either under growth or osteoinductive conditions. During the course of culture, gene expression of alkaline phosphatase (ALP), osteocalcin (OC) and collagen I (COL-I) as well as Smad5 and focal adhesion kinase (FAK), two signal transduction molecules involved in BMP-2 or integrin signaling were analyzed. Furthermore, calcium and collagen I deposition, as well as cell densities and proliferation, were determined using fluorescence microscopy. The incorporation of BMP-2 into PLLA-collagen type I nanofibers resulted in a decrease in diameter as well as pore sizes of the scaffold. Mesenchymal stem cells showed better adherence and a reduced proliferation on BMP-containing scaffolds. This was accompanied by an increase in gene expression of ALP, OC and COL-I. Furthermore the presence of BMP-2 resulted in an upregulation of FAK, while collagen had an impact on the gene expression of Smad5. Therefore these different strategies can be combined in order to enhance the osteoblast differentiation of hMSC on PLLA based nanofiber scaffold. By doing this, different signal transduction pathways seem to be up regulated.