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The healing after rotator cuff surgery is still dissatisfying, and increased muscle fatty infiltration even more impairs the healing success. To achieve sufficient healing after rotator cuff reconstructions, the use of growth factors may be one possibility. The aim of the study was to identify a possible relationship between fatty infiltration of the supraspinatus muscle and cellular biological characteristics and stimulation potential of tenocyte-like cells (TLCs). TLCs of 3 donor groups differing in grade of muscle fatty infiltration were analyzed for their cellular characteristics and were stimulated with BMP-2 or BMP-7 in a 3D scaffold culture. The cell count and potency for self-renewal were significantly decreased in TLCs from donors with high muscle fatty infiltration compared to the lower fatty infiltration groups. Cell count and collagen-I expression as well as protein synthesis were stimulated by growth factors. Interestingly, TLCs of the high fatty infiltration group exhibited a weaker stimulation potential compared to the other groups. TLCs from donors with high muscle fatty infiltration generally revealed inferior characteristics compared to cells of lower fatty infiltration groups, which may be one reason for a weaker healing potential and may represent a possible starting point for the development of future treatment options.

Replacing an infected prosthesis with a bioimplant provides a hopeful alternative in septic vascular surgery. The objective of this study was to determine the effect of fibroblast endothelial growth factors (FGF) and vascular endothelial growth factors (VEGF) coating on a decellularized vascular graft in a rat model and the possible impact on recellularization processes. Rat aortas were decellularized, crosslinked with genipin, and coated with poly-(D, L) lactide containing either FGF or VEGF. Observation periods were 6 and 12 weeks. Surprisingly, we found moderate accumulation of giant cells around the grafts that contained poly-(D, L) lactide acid. FGF and VEGF grafts showed massive stimulation of giant cells and eosinophils leading to complete graft encapsulation (P < 0.05). Pseudointmal hyperplasia was significantly increased in the FGF group (P < 0.05). Both results can only be interpreted as very negative. We achieved a situation in diametric opposition to that which we had hoped for. These data demonstrate that the use of growth factors may produce harmful side effects.

Hyperhomocysteinemia (HHCY) has been linked to fragility fractures and osteoporosis. Folate and vitamin B(12) deficiencies are among the main causes of HHCY. However, the impact of these vitamins on bone health has been poorly studied. This study analyzed the effect of folate and vitamin B(12) deficiency on bone in rats. We used two groups of rats: a control group (Co, n = 10) and a vitamin-deficient group (VitDef, n = 10). VitDef animals were fed for 12 wk with a folate- and vitamin B(12)-free diet. Co animals received an equicaloric control diet. Tissue and plasma concentrations of homocysteine (HCY), S-adenosyl-homocysteine (SAH), and S-adenosyl-methionine (SAM) were measured. Bone quality was assessed by biomechanical testing (maximum force of an axial compression test; F(max)), histomorphometry (bone area/total area; B.Ar./T.Ar.], and the measurement of biochemical bone turnover markers (osteocalcin, collagen I C-terminal cross-laps [CTX]). VitDef animals developed significant HHCY (Co versus VitDef: 6.8 +/- 2.7 versus 61.1 +/- 12.8 microM, p < 0.001) that was accompanied by a high plasma concentration of SAH (Co versus VitDef: 24.1 +/- 5.9 versus 86.4 +/- 44.3 nM, p < 0.001). However, bone tissue concentrations of HCY, SAH, and SAM were similar in the two groups. Fmax, B.Ar./T.Ar., OC, and CTX did not differ between VitDef and Co animals, indicating that bone quality was not affected. Folate and vitamin B(12) deficiency induces distinct HHCY but has no effect on bone health in otherwise healthy adult rats. The unchanged HCY metabolism in bone is the most probable explanation for the missing effect of the vitamin-free diet on bone.

Nitrogen containing bisphosphonates such as zoledronic acid (ZOL) are clinically used to prevent osteoclast induced bone loss. Previous studies indicated that bisphosphonates prevent osteoclast formation, decreases their resorption activity and lead to osteoclast apoptosis. Aim of the study was to investigate the effect of ZOL on fusion and resorption activity of osteoclast like cells (OLC) derived from human peripheral blood mononuclear cells (PBMNC) in vitro. For application of ZOL a local drug delivery system based on a coating for medical devices was used. ZOL was incorporated in the coating based on Poly(D,L-Lactide) (PDLLA) in different concentrations (10–50 μM). Control groups were treated without ZOL or ZOL pure substance in corresponding concentrations. Human PBMNCs were isolated and stimulated to form OLCs. After an experimental period of 144 h, TRAP staining of polynucleated cells was performed and TRAP positive cells were counted. A pit formation assay was performed and resorption lacunas on dentin chips were counted. Results showed a significant dose dependent decrease in the number of TRAP positive cells after exposure to ZOL incorporated in the drug delivery system or applied as pure substance. The amount of resorption lacunas was also dose dependent decreased using both application methods. In conclusion, exposure to specific concentrations of ZOL incorporated in a drug delivery system showed a significant decrease in OLC formation and activity comparable to the effect of pure substance. The effect on osteoclasts might be of clinical benefit to reduce orthopedic implant loosening and to support fracture healing. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007

Pin loosening is a major complication in external fixation. Biological and mechanical conditions play an important role in the maintenance and enhancement of the implant-bone interface in fracture fixation. It is thought that biodegradable coatings may be capable of preventing pin track infection and pin loosening. The goal of this study was therefore to analyze the influence of a biodegradeable coating on the osseous integration of Schanz' screws during fracture treatment. Standardized osteotomies (3-mm fracture gap) of the right tibiae were performed on 16 sheep and stabilized by an AO mono-lateral external fixator. Additional, mechanically less loaded Schanz' screws were also mounted. All screws were randomly coated with biodegradable poly(D,L-lactide). The sheep were sacrificed after 9 weeks. All screws were removed and rolled on blood agar plates for microbiological analysis. Histological sections of the pin tracks were histochemically and morphometrically analyzed. Clinically, no signs of severe infection were visible. Microbiological analysis revealed 14.8% colonization by Staphylococcus aureus in the coated and 29% in the uncoated screws. Histomorphometry of the bone surrounding the Schanz' screws revealed that significantly more osseous integration had occurred on poly(D,L-lactide)-coated screws in the absence of bacterial colonization. Significantly more bone remodeling and a higher osteoclastic activity was seen near the screw-bone interface in the uncoated screw group. Up to a threefold increase in new bone formation and more severe remodeling was observed around the screw entry compared to the pin exit in all groups. Loaded screws showed significantly more callus formation around the exit sites than their less loaded counterparts. In the present study, poly(D,L-lactide) coating of Schanz' screws was found to enhance osseous integration in the absence of bacterial colonization in sheep by causing less cortical remodeling and less osteoclastic activity in the cortices compared to uncoated screws. Additionally, the coating appeared to reduce the instances of pin track infections. Mechanical loading showed an adverse effect on bone formation and remodeling. It has been shown that both biological and mechanical factors play an important role in the maintenance of osseous integrity of the pin-bone interface. Poly(D,L-lactide) coating of Schanz' screws does not prevent osseous destruction and severe bacterial colonization along the pin tracts, but can improve osseous integration of Schanz' screws in the absence of infection.

Schraubenkanal-Infektionen und Schraubenlockerungen sind wesentliche Komplikationen bei der Fixateur externe-Osteosynthese. In dieser Studie wurde der Einfluss einer Poly(D,L-laktid)-Beschichtung auf die ossare Integration der Schrauben wahrend der Frakturversorgung analysiert. An 16 Schafen wurden Osteotomien der rechten Tibia mit einem monolateralen Fixateur externe stabilisiert. Zusatzlich wurden mechanisch unbelastete Schanz' Schrauben eingebracht. Die Halfte der implantierten Schrauben wurde mit Poly(D,L-laktid) beschichtet. Nach neun Wochen wurden die Schrauben entnommen, auf Blutagar-Platten ausgerollt und mikrobiologisch untersucht. Es erfolgte die histologische Aufarbeitung der Pintrakts und eine anschliesende histochemische und histomorphometrische Analyse. Obwohl klinisch keine Anzeichen fur Infektionen beobachtet wurden, zeigte die mikrobiologische Bewertung bei 15 % der beschichteten Schrauben versus 29 % der unbeschichteten Schrauben eine Kontamination durch Staphylococcus aureus. Die histologische Auswertung zeigte, dass beschichtete Schrauben eine bessere Osteointegritat (p=0,0006) als unbeschichtete Implantate aufwiesen. In der Histomorphometrie wurde in der Gruppe der unbeschichteten Schrauben ein hoheres knochernes Remodeling (p = 0,006) und eine hohere Osteoklastenaktivitat (p = 0,019) festgestellt. Belastete Schrauben zeigten mehr Kallus am Schraubenaustritt (p = 0,048). Eine Poly(D,L-laktid)-Beschichtung bei Schanz' Schrauben scheint die ossare Integration durch eine Verminderung des kortikalen Remodelings und der Osteoklastenaktivitat zu verbessern. Improvement of bony integration of Schanz’ screws by means of a Poly(D,L-laktid) coating Pin track infections and pin loosening are major complications in fracture treatment by means of external fixation. In this study, the influence of Poly(D,L-lactid)-coating on osseous integration of Schanz screws was analysed during bone healing. In sixteen sheep, osteotomies of the right tibia were stabilized by means of monolateral external fixators. In addition, mechanically unloaded Schanz' screws were inserted. One half of the implanted screws was coated with Poly(D,L-lactid). After nine weeks in vivo, the screws were extracted, rolled on blood agar plates and microbiologically analysed, followed by histological, immunhistochemical and histomorphometrical analyses of the pin tracks. Even though no clinical signs of infection were observed, micro biological evaluation indicated a contamination by Staphylococcus aureus of 15 % in the coated screws and of 29 % in the uncoated screws. The histological analyses showed an improved osseous interagtion in the coated screws (p=0,0006) compared to the uncoated implants. In histomorphometry, the group of the uncoated screws showed an increased bone remodelling (p = 0,006) and an increased osteoclast activity (p = 0,019). Mechanically loaded screws had increased callus formation at the pin exit site (p = 0,048). The poly(D,L-lactid) coating of Schanz' screws seems to enhance the osseous integration by reducing cortical remodelling and osteoclast activity.

Infection related to osteosynthesis often has dramatic consequences for the patient. Prolonged hospitalization with systemic antibiotic therapy, several revision procedures, possible amputation, and even death may occur. To investigate the pathology of infection in orthopedic surgery, a new rat model of implant related osteomyelitis was developed. Three different concentrations (10(6), 10(3), and 10(2) colony-forming units (CFU)/10 microl) of Staphylococcus aureus were inoculated into the tibial medullary cavity with simultaneous insertion of a titanium Kirschner wire. Controls received phosphate-buffered saline (PBS). Each group consisted of 10 animals. Animals were followed for 4 weeks until sacrifice. X-rays of the tibiae were taken weekly, blood counts were analyzed, and body temperature and weight were determined. After sacrifice, infection was evaluated by histological and microbiological investigations. All animals inoculated with Staph. aureus in either concentration developed microbiological, histological, and radiological signs of osteomyelitis in correlation to the amount of inoculated bacteria. X-rays clearly revealed osseous destruction after 14 days with progression of osteomyelitis during the following weeks. CFU/g bone and bone weight after sacrifice showed dependence on the amount of inoculated CFU. The histological results confirmed the radiological findings. No significant changes in blood counts, body weight, and body temperature between the groups could be observed. The results demonstrate that it is possible to develop a model of implant-related osteomyelitis in rats with dependence on the amount of inoculated bacteria. No other promoters of infection besides intramedullary insertion of titanium Kirschner wires were used in this model.

Growth factors IGF-I and TGF-beta1 are known to stimulate fracture healing. The purpose of this study was to investigate the role of locally applied IGF-I and TGF-beta1 during the early phase of fracture healing (Days 5, 10, and 15 after fracture) on cellular processes like proliferation and differentiation in a rat model. Two different immunohistochemical markers were used to analyze cell proliferation: (1) injection of the thymidine analogue BrdU and subsequent immunohistochemical staining for BrdU-positive nuclei, and (2) the antibody against the "proliferating cell nuclear antigen" (PCNA). In comparison, both methods revealed similar results concerning the types of proliferating cells at the different time points and the two groups. Labeling indices of both methods showed very good correlation (e.g., r(s): 0.887 and p < 0.001 at day 10 in the control group without growth factors). Comparison of the callus morphology and the proliferation rate showed differences during fracture healing due to the local application of IGF-I and TGF-beta1 from coated implants. At Day 5 the callus of the group treated with growth factors displayed an earlier appearance of cartilage compared to the control group. This was accompanied by an onset of cell proliferation in chondrocytes. Likewise, at the later time points an enhanced maturation of the callus tissue and the proliferation pattern were detectable in the growth-factor group. These results indicate that local application of IGF-I and TGF-beta1 accelerates early cellular processes during fracture healing.

Previous in vivo studies revealed a stimulating effect of locally applied IGF-I and TGF-β1 released from poly(D,L-lactide)-coated titanium implants on rat and porcine fracture healing. The purpose of the present study was to evaluate the effect of IGF-I (5% w/w) and TGF-β1 (1% w/w) and the carrier PDLLA on osteoblasts in cell culture to improve the understanding of these growth factors. The well-characterized human osteoblast cell line hFOB 1.19 was used in the study. The implants and cells were cocultured in a noncontact manner. The cells were incubated for 10 days in total, and the implants (n = 6 each group and time point) were added for 1 h, 12 h, 24 h, 2 d, 4 d, or 10 d. To analyze a possible effect of the growth factors or the coating, cell proliferation, metabolism, and differentiation were investigated. As an indicator for differentiation the production of collagen I was chosen. All experimental groups showed comparable cell vitality. No change in the pH of the medium was detectable between the analyzed groups. When the effect of the titanium implant and the PDLLA coating were compared with the control culture, no differences in proliferation, metabolic activity, and collagen I production were detectable. The osteoblasts treated with IGF-I and TGF-β1 released from PDLLA revealed a significantly enhanced collagen I production with a decrease in proliferation and metabolic activity compared to the other groups. No significant differences in collagen I production were seen due to the incubation time points. None of the experimental groups evoked an immunological response on mouse macrophages. In conclusion, the PDLLA-carrier showed no negative effect on osteoblasts, whereas the incorporated growth factors stimulated osteoblast differentiation. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 65B: 157–162, 2003

Hydroxyapatite (HAP) is widely used as an osteoconductive coating for orthopedic implants. So far standard coating methods like plasma spraying produce a relatively thick coating layer (>30 μm). In addition, the chemical structure of the HAP may be altered because of the heating throughout the coating process. This may have negative effects on the coating stability, implant fixation, and induction of bone formation. The relatively thick layer may detach from the implant with the risk of wear debris. In the present study the potential of a newly developed HAP coating of implants on osteointegration was investigated in a rat model. The coating method, based on an electrochemical process, is applied in a graded manner and results in a biodegradable HAP coating with a thickness of approximately 2 μm. Coated versus uncoated titanium Kirschner wires (1.4-mm diameter) were inserted into the medullary cavity of the right femora of 5-month old female Sprague Dawley rats (n=36) in a retrograde fashion. Throughout an experimental period of 2 months the osteointegration was traced radiologically. After this time the animals were sacrificed and the implant integration was tested biomechanically with the use of a push-out test. To analyze the bone-implant interface, histological sections (80 μm) were investigated with an image analyzing system. The biomechanical testing revealed a significantly higher implant fixation in the group treated with the HAP-coated implant (shear strength: 27.8 ± 6.7 MPa) compared to control (shear strength: 8.08 ± 3.4 MPa). The histological analyses demonstrated a better ingrowth of the implants in the HAP group with significantly more direct bone-implant contacts compared to the control group. The results demonstrate that the HAP coating promotes implant osteointegration in a rat model. © John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 63: 168–172, 2002; DOI 10.002/jbm.10130

Local application of growth factors like insulin like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta1) from a biodegradable thin layer of poly(D,L-lactide) (PDLLA) coated implants could stimulate fracture healing. A new "cold coating technique" for metallic implants was established to produce a biodegradable coating with a high mechanical stability that provides a continuous release of incorporated growth factors. The properties of this bioactive coating were investigated in vitro and in vivo. Scanning electron microscope analysis revealed a coating thickness of in average 14.8 microm on titanium and 10.7 microm on steel wires. Intramedullary implantation and extraction experiments depicted a loss of PDLLA coating from titanium and steel implants of less than 5%. After explantation of the implants, the coating displayed a complete and regular layer without any defects of PDLLA uncovering the metallic surface. Smear tests demonstrate that the coating can be performed under sterile conditions. The PDLLA depicted a reduction of about 8% within 6 weeks in vitro and in vivo. The growth factors were incorporated in a stable form and demonstrated a loss of stability of less than 3% within 42 days and less than 5% within one year. In an elution experiment, 54% IGF-I and 48% TGF-beta1 were released within the first 48 h. After 42 days, 76% of IGF-I and 71% of TGF-beta1 were detected in the elution fluid by ELISA. Comparable results were obtained in the in vivo experiments after 42 days.

Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO-induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae.

This article investigates a method of modifying and optimizing the biocompatibility of decellularized vascular bioimplants when treated with a specialized, drug eluting coating. For this purpose, we carried out aortic transplantations using a porcine model. Decellularized, cross-linked aortic grafts were coated with poly(D,L-lactide) (PDLLA). To this coating, we added the anticoagulant drug lepirudin which, following transplantation, would be linearly eluted. These aortic grafts are easily manipulated in surgery. It was shown that, as a result of the lepirudin-eluting coating, the rate of thrombogenesis was reduced and the patency rate was significantly improved. However, lumen-stenosing pseudointima developed in all of the transplants and was not effected by PDLLA coating. Furthermore, no evidence of recellularisation was documented. This trial demonstrates that polymer coating of decellularized tissue is possible. Neointimal hyperplasia and the absence of cellular repopulation mark the negative consequences of this concept.