Jürgen R. J. Paletta

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.

Influence of Poly(L-Lactic Acid) Nanofibers and BMP-2–Containing Poly(L-Lactic Acid) Nanofibers on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells

The aim of this study was to characterize synthetic poly-(L-lactic acid) (PLLA) nanofibers concerning their ability to promote growth and osteogenic differentiation of stem cells in vitro, as well as to test their suitability as a carrier system for growth factors. Fiber matrices composed of PLLA or BMP-2–incorporated PLLA were seeded with human mesenchymal stem cells and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of alkaline phosphatase (ALP), osteocalcin (OC), and collagen I (COL-I). Furthermore, COL-I and OC deposition, as well as cell densities and proliferation, were analyzed using fluorescence microscopy. Although the presence of nanofibers diminished the dexamethasone-induced proliferation, there were no differences in cell densities or deposition of either COL-I or OC after 22 days of culture. The gene expression of ALP, OC, and COL-I decreased in the initial phase of cell cultivation on PLLA nanofibers as compared to cover slip control, but normalized during the course of cultivation. The initial down-regulation was not observed when BMP-2 was directly incorporated into PLLA nanofibers by electrospinning, indicating that growth factors like BMP-2 might survive the spinning process in a bioactive form.

Characterization of a PLLA-collagen I blend nanofiber scaffold with respect to growth and osteogenic differentiation of human mesenchymal stem cells.

The aim of this study was to enhance synthetic poly(L-lactic acid) (PLLA) nanofibers by blending with collagen I (COLI) in order to improve their ability to promote growth and osteogenic differentiation of stem cells in vitro. Fiber matrices composed of PLLA and COLI in different ratios were characterized with respect to their morphology, as well as their ability to promote growth of human mesenchymal stem cells (hMSC) over a period of 22 days. Furthermore, the course of differentiation was analyzed by gene expression of alkaline phosphatase (ALP), osteocalcin (OC), and COLI. The PLLA-COLI blend nanofibers presented themselves with a relatively smooth surface. They were more hydrophilic as compared to PLLA nanofibers alone and formed a gel-like structure with a stable nanofiber backbone when incubated in aqueous solutions. We examined nanofibers composed of different PLLA and COLI ratios. A composition of 4:1 ratio of PLLA:COLI showed the best results. When hMSC were cultured on the PLLA-COLI nanofiber blend, growth as well as osteoblast differentiation (determined as gene expression of ALP, OC, and COLI) was enhanced when compared to PLLA nanofibers alone. Therefore, the blending of PLLA with COLI might be a suitable tool to enhance PLLA nanofibers with respect to bone tissue engineering.

Grice-Green Procedure for Severe Hindfoot Valgus in Ambulatory Patients with Cerebral Palsy

Grice-Green extra-articular subtalar arthrodesis is a treatment option for advanced valgus rearfoot deformity of neuromuscular origin in young patients. The purpose of this study was to evaluate long-term results of the procedure in ambulatory cerebral palsy patients. From January 1975 to December 1993, 57 operations were performed in 39 patients (22 males, 17 females) with a mean age of 7.8 ± 2.7 (range 3.9-14.4) years for excessive symptomatic hindfoot valgus. Thirty-five patients (51 feet) were followed for a mean 22.6 ± 4.6 (range 16.0-32.3) years. Preoperative GMFCS score, ambulation, hindfoot position, podoscopic view of the weight-bearing feet, and radiological examinations, along with complications and orthotic use, were compared. Based on a clinical rating scale, 39 results were excellent or good, 8 were fair, and 4 were poor. The poor results were attributed to hindfoot valgus recurrence or varus overcorrection. One case needed revisional surgery owing to slippage of the graft. Overall, the GMFCS score, level of ambulatory distance, and use of foot orthoses improved. Grice-Green subtalar arthrodesis did not result in early degenerative changes at the midterm follow-up. The procedure offers safe and long-lasting correction for severe and symptomatic hindfoot disorders in patients with cerebral palsy. Together with physiotherapy and orthotic use, it can improve ambulation. Whereas a slight hindfoot valgus in patients with cerebral palsy is tolerable without lack of function, overcorrection should be avoided.

The role of mesenchymal stem cells in the pathogenesis of Co–Cr–Mo particle induced aseptic loosening: An in vitro study

Aseptic loosening is the major factor of failed arthroplasty. Among several theories the particle disease theory is commonly accepted. Different studies examined the complex interactions between wear debris and surrounding cells, especially the monocytic and osteoblastic lineage. This study was designed to elucidate the impact of cobalt-chromium-molybdenum (Co-Cr-Mo) particles on the osteoblastic differentiation and proliferation of human mesenchymal stem cells (hMSC), with respect to the disease pattern of aseptic loosening. The hMSC were incubated in the presence of Co-Cr-Mo particles in different concentrations under growth and osteoinductive conditions. Obtained cultures were analyzed, with respect to cell density and proliferation, using CASY cell count system and Ki-67 immunostaining. Osteogenic differentiation was analyzed by fluorescence microscopy using antibodies for collagen I, alcaline phosphatase, osteocalcin and osteopontin. Additionally, scanning electron microscopy was used to analyze the localisation of Co-Cr-Mo particles in the culture system. Our findings indicate that these particles were located within the hMSC. Proliferation, as well as cell density, was diminished. The remaining cells showed increased staining of osteocalcin and osteopontin, with visible differences in deposition of these proteins, indicating a deregulation of matrix formation and differentiation respectively. Therefore, it is likely that this influence of Co-Cr-Mo particles on hMSC are involved in the disease pattern of aseptic loosening.

Lack of Obvious Influence of PLLA Nanofibers on the Gene Expression of BMP-2 and VEGF during Growth and Differentiation of Human Mesenchymal Stem Cells

Growth factors like bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) play an important role in bone remodeling and fracture repair. Therefore, with respect to tissue engineering, an artificial graft should have no negative impact on the expression of these factors. In this context, the aim of this study was to analyze the impact of poly(L-lactic acid) (PLLA) nanofibers on VEGF and BMP-2 gene expression during the time course of human mesenchymal stem cell (hMSC) differentiation towards osteoblasts. PLLA matrices were seeded with hMSCs and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of VEGF and BMP-2. Furthermore, BMP-2–enwoven PLLA nanofibers were used in order to elucidate whether initial down-regulation of growth factor expression could be compensated. Although there was a great interpatient variability with respect to the expression of VEGF and BMP-2, PLLA nanofibers tend to result in a down-regulation in BMP-2 expression during the early phase of cultivation. This effect was diminished in the case of VEGF gene expression. The initial down-regulation was overcome when BMP-2 was directly incorporated into the PLLA nanofibers by electrospinning. Furthermore, the incorporation of BMP-2 into the PLLA nanofibers resulted in an increase in VEGF gene expression. Summarized, the results indicate that the PLLA nanofibers have little effect on growth factor production. An enhancement in gene expression of BMP-2 and VEGF can be achieved by an incorporation of BMP-2 into the PLLA nanofibers.

Influence of Synovial Fluid on Human Osteoblasts: An In Vitro Study

Osseous graft healing at the tendon bone interface after anterior cruciate ligament (ACL) reconstruction is unsatisfactory in 10—25%, depending on the evaluation criteria or the kind of graft used for reconstruction. Mechanical as well as biological aspects are currently discussed. Since osteoblasts play an important role in the osseous integration of an ACL graft, we hypothesize that synovial fluid (SF), when entering the bone tunnel, has an inhibitory effect on osteoblasts. In order to verify this hypothesis, human osteoblasts (p3) were incubated in the presence of SF or partially purified SF. Proliferation was assayed using MTT or BrdU assay. Gene expression of osteoblast markers (alkaline phosphatase, collagen I, and osteocalcin) were determined by TaqMan analysis. In the control group, SF was exchanged by fetal calf serum (FCS). The results showed osteoblast proliferation in the presence of SF as well as in partially purified heat-pretreated synovial fluid. Native SF induced alkaline phosphatase and collagen I gene expression. No induction of the osteocalcin gene was observed in the experiment. These results were comparable to that obtained with FCS. These findings suggest that SF stimulated proliferation of osteoblasts in vitro. This effect is mediated, in part, by heat-stable components of SF. In addition, the expression of osteoblast marker genes alkaline phosphatase and collagen I, but not osteocalcin, was induced by SF. Therefore, problems associated with cruciate ligament reconstruction might be due to the inhibition of osteoblast differentiation. If so, this is not a specific attribute of SF, but also applies to serum.

Influence of poly-(L-lactic acid) nanofiber functionalization on maximum load, Young's modulus, and strain of nanofiber scaffolds before and after cultivation of osteoblasts: an in vitro study.

The aim of this study was to characterize the influence of functionalization of synthetic poly-(L-lactic acid) (PLLA) nanofibers on mechanical properties such as maximum load, elongation, and Youngs modulus. Furthermore, the impact of osteoblast growth on the various nanofiber scaffolds stability was determined. Nanofiber matrices composed of PLLA, PLLA-collagen, or BMP-2–incorporated PLLA were produced from different solvents by electrospinning. Standardized test samples of each nanofiber scaffold were subjected to failure protocol before or after incubation in the presence of osteoblasts over a period of 22 days under osteoinductive conditions. PLLA nanofibers electrospun from hexafluoroisopropanol (HFIP) showed a higher strain and tended to have increased maximum loads and Youngs modulus compared to PLLA fibers spun from dichloromethane. In addition, they had a higher resistance during incubation in the presence of cells. Functionalization by incorporation of growth factors increased Youngs modulus, independent of the solvent used. However, the incorporation of growth factors using the HFIP system resulted in a loss of strain. Similar results were observed when PLLA was blended with different ratios of collagen. Summarizing the results, this study indicates that different functionalization strategies influence the mechanical stability of PLLA nanofibers. Therefore, an optimization of nanofibers should not only account for the optimization of biological effects on cells, but also has to consider the stability of the scaffold.

Liposarcoma of the forearm in a man with type 1 neurofibromatosis: a case report

IntroductionThe combination of neurofibromatosis and liposarcoma is very rare. We present a case of a dedifferentiated liposarcoma in the forearm, as a complication in a patient with neurofibromatosis type 1.Case presentationA Caucasian man with neurofibromatosis type 1 presented at our clinic complaining of a slow growing swelling on his left forearm over a period of one and a half years. Clinical examination and history pointed to malignancy. Radiological examination inclusive of magnetic resonance imaging and positron emission tomography confirmed our suspicion. A final diagnosis of dedifferentiated high-grade liposarcoma with axillary lymph node metastases was established after a pathological examination of a tumour biopsy. The consulting tumour board recommended either an elbow exarticulation or an accurate radical local resection including the metastatic axillary lymph nodes. Fortunately, we were able to perform an R-zero resection and the forearm could be saved. The treatment was completed with postoperative radiotherapy of the left forearms operative bed, the left axillary and the supraclavicular regions. The patient decided against adjuvant chemotherapy.ConclusionLiposarcoma complicating neurofibromatosis type 1 is a very rare combination. Up to now, only five cases have been reported in the literature. We are adding a new case to this short list to stress the importance of early recognition. It is the first known case with this disease combination in an upper extremity. Liposarcoma is usually treated by surgery followed by radiotherapy. The role of chemotherapy is controversial and should be based on a decision made on a case-by-case basis.

Results of titanium locking plate and stainless steel cerclage wire combination in femoral fractures

Background: Some in vitro studies warn combining different metals in orthopedic surgery. The aim of this study is to determine the impact of combining titanium and stainless steel on bone healing and the clinical course of patients undergoing internal fixation of femoral fractures. Materials and Methods: 69 patients with femoral fractures had polyaxial locking plate osteosynthesis. The locking plate was made of a titanium alloy. Two different cohorts were defined: (a) sole plating and (b) additional stainless steel cerclage wiring. Postoperative radiographs and clinical followup were performed at 6 weeks, 3 months and 12 months. Results: Cohorts A and B had 36 and 33 patients, respectively. Patient demographics and comorbidities were similar in both groups. In two cases in cohort A, surgical revision was necessary. No complication could be attributed to the combination of titanium and stainless steel. Conclusion: The combination of stainless steel cerclage wires and titanium plates does not compromise fracture healing or the postoperative clinical course.