Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice.

BACKGROUND: Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT). METHODS: Vertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM). RESULTS: Bone of female α7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N(+) (p < 0.05) and C4H8N(+) (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM. CONCLUSIONS: Our results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7ß2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and ß-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.

Arthroscopic treatment of post-traumatic shoulder instability in children and adolescents.

Traumatic dislocation of the shoulder in childhood is rare, accounting for only 0.01% of all injuries in this age class. However, the literature does contain indications that post-traumatic instabilities and redislocations occur with more than average frequency in children and adolescents. This study describes the technique and results of arthroscopic stabilization of the shoulder joint before the completion of growth. Over a period of 4 years, seven patients were treated prior to reaching skeletal maturity. Five patients were treated by means of arthroscopically assisted labral refixation using suture anchor systems. Clinical follow-up was undertaken after between 6 and 48 months using a modified Constant-Murley score. Instabilities, re-dislocations and postoperative impairment of mobility did not occur after arthroscopic treatment, with a score of 92 points. Arthroscopic labral refixation is a reliable, surgical treatment procedure for post-traumatic shoulder instability in adults. This study showed that it can be used successfully in children and young people prior to skeletal maturity. No alteration of the surgical procedure of Bankart repair in adults is necessary. Considerable experience in adult shoulder reconstruction promises excellent results in children and adolescents, too. There is no evidence of growth disturbance or biodegradation associated problems in this group of children and adolescents. Delay of labral refixation until adulthood is therefore not indicated.

Expression of non-neuronal cholinergic system in osteoblast-like cells and its involvement in osteogenesis.

Acetylcholine (ACh) is detected in a variety of non-neuronal cells where it acts as a para/autocrine signaling molecule controlling basic cell functions such as proliferation, differentation, and maintenance of cell-cell contacts. ACh-synthesizing enzymes include choline acetyltransferase and carnitine acetyltransferase (CarAT). ACh is released through vesicular exocytosis or directly from the cytoplasm via organic cation transporters (OCT). Extracellular ACh binds to nicotinic (nAChR) and muscarinic receptors (MR). Degradation of ACh is performed by acetylcholinesterase and butyrylcholinesterase (BChE). Here, we have determined whether these molecules are expressed in osteoblast-like cells, by means of reverse transcription polymerase chain reaction and immunohistochemistry, focusing on nAChR subunits alpha3 and alpha5. RNA for CarAT, OCT-1, M2R, M5R, nAChR subunits alpha3, alpha5, alpha9, alpha10, beta2, beta3, and BChE were detected in human (SAOS-2) and murine (MC3T3-E1) osteoblast-like cells. Other cholinergic components were only expressed species-specifically, e.g., M3R and nAChR subunit alpha7. Immunhistochemistry localized the nAChR subunits alpha3 and alpha5 in osteoblasts in vitro and in vivo where they were up-regulated after application of bone morphogenetic protein-2 (BMP-2) during fracture healing in a rat model. Thus, the cholinergic system of osteoblast-like cells might be regulated by BMP-2 during bone remodeling. Osteoblast-like cells express all necessary enzymes, transporters, and receptors for ACh synthesis and recycling.

Observations on the microvasculature of bone defects filled with biodegradable nanoparticulate hydroxyapatite.

The microvascularization of metaphyseal bone defects filled with nanoparticulate, biodegradable hydroxyapatite biomaterial with and without platelet factors enrichment was investigated in a minipig model. Results from morphological analysis and PECAM-1 immunohistochemistry showed the formation of new blood vessels into the bone defects by sprouting and intussusception of pre-existing ones. However, no significant differences were observed in the microvascularization of the different biomaterials applied (pure versus platelet factors-enriched hydroxyapatite), concerning the number of vessels and their morphological structure at day 20 after operation. The appearance of VEGFR-2 positive endothelial progenitor cells in the connective tissue between hydroxyapatite particles was also found to be independent from platelet factors enrichment of the hydroxyapatite bone substitute. In both groups formation of lymphatic vessels was detected with a podoplanin antibody. No differences were noted between HA/PLF- and HA/PLF+ implants with respect to the podoplanin expression level, the staining pattern or number of lymphatic vessels. In conclusion, the present study demonstrates different mechanisms of blood and lymphatic vessel formation in hydroxyapatite implants in minipigs.

[Bond strength of a bioresorbable bone adhesive: results of a biomechanical study in a sheep model]. / Biomechanische Analysen zur Verbundfestigkeit eines bioresorbierbaren Knochenklebers: experimentelle Untersuchungen am Schafsmodell.

The purpose of this study was to investigate the bond strength of a new bone adhesive based on ethylene glycol-oligolactide-bismethacrylate on 36 sheep. A 2-cm metaphysial segment was produced on the ulna of each sheep by an oscillating saw and it was not stabilized by any type of additional osteosynthesis. Adhesive was applied to the osteotomy gaps in 18 sheep, the remaining 18 animals served as controls. A total of 6 animals with glue and 6 controls were euthanized after 21, 42 and 84 days. The bond strength after repair of the gaps through bone adhesive compared to a control group was studied by using a four-point bending test. There was a continual increase of bending stiffness from 21 to 84 days in all sheep, with the highest bending stiffness of 102.83 N/mm2 by the glue group after 84 days as opposed to the control group with 58.48 N/mm2 (p = 0.25). Morphological investigations showed more callus formation by the control group than the adhesive group after 84 days (p = 0.04). In addition, an in vitro gluing of the ulna segment was performed with a four-point bending test after 10, 60 and 360 min polymerization time. The in vitro gluing of the ulna segment showed a continual increase of bending stiffness to 17.32 N/mm2 after 360 min (p = 0.59).

Connexin 43 expression of foreign body giant cells after implantation of nanoparticulate hydroxyapatite.

In bone a role of connexin 43 has been implicated with the fusion of mononuclear precursors of the monocyte/macrophage lineage into multinucleated cells. In order to investigate the putative role of connexin 43 in formation of bone osteoclast-like foreign body giant cells which are formed in response to implantation of biomaterials, nanoparticulate hydroxyapatite had been implanted into defects of minipig femura. After 20 days the defect areas were harvested and connexin 43 expression and synthesis were investigated by using immunohistochemistry, Western Blot, and in situ hybridization within macrophages and osteoclast-like foreign body giant cells. Morphological analysis of gap junctions is performed ultrastructurally. As shown on protein and mRNA level numerous connexin 43 positive macrophages and foreign body giant cells (FBGC) were localized within the granulation tissue and along the surfaces of the implanted hydroxyapatite (HA). Besides, the formation of FBGC by fusion of macrophages could be shown ultrastructurally. Connexin 43 labeling observed on the protein and mRNA level could be attributed to gap junctions identified ultrastructurally between macrophages, between FBGC, and between FBGC and macrophages. Annular gap junctions in the cytoplasm of FBGC pointed to degradation of the channels, and the ubiquination that had occurred in the course of degradation was confirmed by Western blot analysis. All in all, the presently observed pattern of connexin 43 labeling refers to an functional role of gap junctional communication in the formation of osteoclast-like foreign body giant cells formed in response to implantation of the nanoparticulate HA.

Enhanced alpha 1(I) mRNA expression in frozen shoulder and dupuytren tissue.

The purpose of this study has been to investigate collagen I and III synthesis during the fibrosing stage of frozen shoulder and Dupuytren samples in comparison to normal capsule tissue. - By using the quantitative PCR significantly increased levels of alpha 1(I) mRNA transcription in samples of frozen shoulder (p = 0.016) and Duypuytren (p = 0.041) could be demonstrated, whereas alpha 2(I) and alpha 1(III) chains have shown the same mRNA levels as in normal capsule tissue. - Despite an enhancement of alpha 1(I) mRNA transcription in frozen shoulder and Dupuytren samples the intracellular precursor procollagen I and extracellular mature collagen I was detected immunohistochemically in reduced levels. - The structural alteration of collagen I assembly might be caused by disturbed post-translation from the polypeptide chains into the triple helices procollagen I though alpha 1(I) mRNA transcription was significantly increased and alpha 2(I) mRNA transcription was in normal range. Fibroblasts might release high quantities of free alpha 1(I) polypeptide chains or (alpha 1(I)) 3 homotrimer into the extracellular space during the fibrosing stage of frozen shoulder and Dupuytren disease. - In all samples neither differences of alpha 1(III) mRNA transcription nor differences of immunohistochemical staining intensity of collagen III could be seen. This might result from apoptosis of myofibroblasts in the final phase of the fibrosing processes. - The stimulating effect of insulin-like growth factor type I (IGF-I) to induce fibrosis in connective tissue such as scarlet is known. In all patients suffering from frozen shoulder and Dupuytren disease the serum IGF-I level was in a normal range and the IGF-I receptor - (IGFR-I) mRNA transcription in the samples was also in the same level compared with normal capsule tissue.

Human reaming debris: a source of multipotent stem cells.

The biological characteristics of human reaming debris (HRD) generated in the course of surgical treatment of long bone diaphyseal fractures and nonunions are still a matter of dispute. Therefore, the objective of the present investigation has been to characterize the intrinsic properties of human reaming debris in vitro. Samples of reaming debris harvested from 12 patients with closed diaphyseal fractures were examined ultrastucturally and were cultured under standard conditions. After a lag phase of 4-7 days, cells started to grow out from small bone fragments and established a confluent monolayer within 20-22 days. The cells were characterized according to morphology, proliferation capacity, cell surface antigen profile, and differentiation repertoire. The results reveal that human reaming debris is a source of multipotent stem cells which are able to grow and proliferate in vitro. The cells differentiate along the osteogenic pathway after induction and can be directed toward a neuronal phenotype, as has been shown morphologically and by the expression of neuronal markers after DMSO induction. These findings have prompted interest in the use of reaming debris-derived stem cells in cell and bone replacement therapies.

The tissue response to an alkylene bis(dilactoyl)-methacrylate bone adhesive.

Gluing is an attractive technique to fix small bone fragments. However, to date no bone adhesive could be established successfully for all day clinical use. The purpose of this experimental study was to investigate the biocompatibility of a new bone glue based on alkylene bis(dilactoyl)-methacrylate in 36 rabbits. Monocondylar osteotomy of the distal femur was performed and bone glue was applied into the osteotomy gap in 24 rabbits. The remaining 12 animals served as controls. In all rabbits the osteotomy was subsequently stabilized by K-wire osteosynthesis. Six animals of the glue group and 3 controls were euthanized after 7, 21, 42, and 84 days, respectively. Fracture healing and degradation pattern of the glue was studied using histological, histomorphometrical, scanning electron microscopical, and radiological methods. Good resorption of the glue by mononuclear and multinucleated giant cells without prolonged inflammatory processes was observed in the glue group. Histomorphometrical analysis did not reveal any significant differences in fracture healing between the glue and control group at any time. Complete remodelling of the former osteotomy gap was found in all rabbits after 84 days. This bioresorbable bone adhesive exhibited good biocompatibility and its degradation did not interfere with physiological fracture healing.

Plasma transglutaminase factor XIII induces microvessel ingrowth into biodegradable hydroxyapatite implants in rats.

Coagulation factor XIII is a member of the transglutaminase-family. Transgluaminases cross-link either fibrin monomers in blood coagulation or extracellular proteins in extracellular matrix formation. In early stages of bone healing migration and proliferation of endothelial cells lead to formation of new vessels. The aim of this study was to investigate the angiogenetic activity of plasma factor XIII in bone defects filled with nanoparticulate hydroxyapatite paste. A critical size defect was created in the tibial head of rats which was not filled in group I. In group II the defect was filled with hydroxyapatite paste, and in group III with hydroxyapatite paste enriched with factor XIII. Ten days after surgery angiogenesis in the defects was assessed using immunohistochemistry and confocal laser scanning microscopy. Ac16 antibody was used to detect activation of factor XIII into factor XIIIA. In defects without biomaterial (group I) vessel-rich connective tissue and diffuse distribution of capillaries was observed. In defects filled with pure hydroxyapatite (group II) formation of capillaries was limited to the host bone-hydroxyapatite interface. In contrast, addition of plasma factor XIII to hydroxyapatite (group III) stimulated formation of vessels within the biomaterial. The current study reveals that factor XIII can improve angiogenesis in hydroxyapatite.

Glycerol-l-lactide coating polymer leads to delay in bone ingrowth in hydroxyapatite implants.

Glycerol-l-lactide as coating polymer for the delivery of basic fibroblast growth factor (bFGF) from hydroxyapatite (HA) ceramic implants was shown to lead to significant delay in bone ingrowth into the implants compared to implants without the coating polymer. The purpose of this work was to study bone ingrowth in HA ceramic implants with and without the coating polymer but without growth factors to enable differentiation between a locking effect of the pores by the polymer and the fact of inactivation of the growth factors by the polymer, which could both be possible for the delay. A defect was created in the subchondral region of both femurs in 24 miniature-pigs and was either filled by the HA implants with or without the coating polymer. Histomorphometry showed a significant delay in bone ingrowth in the polymer coated implants both after 6 and 12 weeks. Detailed histology revealed that the HA pores were completely "locked" by the polymer leading to complete loss of the osteoconductive properties of the HA. Also electron microscopy showed filling of the HA pores by the polymer. Therefore, it can be concluded that glycerol-l-lactide should not be used to coat HA ceramic implants due to significant delay in bone ingrowth.

Impact of acetylcholine and nicotine on human osteoclastogenesis in vitro.

Recent studies showed that the non-neuronal cholinergic system (NNCS) is taking part in bone metabolism. Most studies investigated its role in osteoblasts, but up to now, the involvement of the NNCS in human osteoclastogenesis remains relatively unclear. Thus, aim of the present study was to determine whether the application of acetylcholine (ACh, 10(−4) M), nicotine (10(−6) M), mineralized collagen membranes or brain derived neurotrophic factor (BDNF, 40 ng/mL) influences the mRNA regulation of molecular components of the NNCS and the neurotrophin family during osteoclastogenesis. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of young healthy donors (n = 8) and incubated with bone fragments and osteoclast differentiation media for 21 days. All the results are based on the measurement of RNA. Real-time RT-PCR analysis demonstrated a down-regulation of nicotinic acetylcholine receptor (nAChR) subunit α2 and muscarinic acetylcholine receptor (mAChR) M3by osteoclastogenesis while BDNF mRNA expression was not regulated. Application of ACh, nicotine, BDNF or collagen membranes did not affect osteoclastic differentiation.No regulation was detected for nAChR subunit α7, tropomyosin-related kinase receptor B (TrkB), and cholineacetyl transferase (ChAT). Taken together, we assume that the transcriptional level of osteoclastogenesis of healthy young humans is not regulated by BDNF, ACh, and nicotine. Thus, these drugs do not seem to worsen bone degradation and might therefore be suitable as modulators of bone substitution materials if having a positive effect on bone formation.

Bone ingrowth in bFGF-coated hydroxyapatite ceramic implants.

This experimental study was performed to evaluate angiogenesis, bone formation, and bone ingrowth in response to osteoinductive implants of bovine-derived hydroxyapatite (HA) ceramics either uncoated or coated with basic fibroblast growth factor (bFGF) in miniature pigs. A cylindrical bone defect was created in both femur condyles of 24 miniature pigs using a saline coated trephine. Sixteen of the 48 defects were filled with HA cylinders coated with 50 microg rhbFG, uncoated HA cylinders, and with autogenous transplants, respectively. Fluorochrome labelled histological analysis, histomorphometry, and scanning electron microscopy were performed to study angiogenesis, bone formation and bone ingrowth. Complete bone ingrowth into bFGF-coated HA implants and autografts was seen after 34 days compared to 80 days in the uncoated HA group. Active ring-shaped areas of fluorochrome labelled bone deposition with dynamic bone remodelling were found in all cylinders. New vessels could be found in all cylinders. Histomorphometric analysis showed no difference in bone ingrowth over time between autogenous transplants and bFGF-coated HA implants. The current experimental study revealed comparable results of bFGF-coated HA implants and autogenous grafts regarding angiogenesis, bone synthesis and bone ingrowth.

BDNF and its TrkB receptor in human fracture healing.

Fracture healing is a physiological process of repair which proceeds in stages, each characterized by a different predominant tissue in the fracture gap. Matrix reorganization is regulated by cytokines and growth factors. Neurotrophins and their receptors might be of importance to osteoblasts and endothelial cells during fracture healing. The aim of this study was to examine the presence of brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) during human fracture healing. BDNF and TrkB were investigated in samples from human fracture gaps and cultured cells using RT-PCR, Western blot, and immunohistochemistry. Endothelial cells and osteoblastic cell lines demonstrated a cytoplasmic staining pattern of BDNF and TrkB in vitro. At the mRNA level, BDNF and TrkB were expressed in the initial and osteoid formation phase of human fracture healing. In the granulation tissue of fracture gap, both proteins--BDNF and TrkB--are concentrated in endothelial and osteoblastic cells at the margins of woven bone suggesting their involvement in the formation of new vessels. There was no evidence of BDNF or TrkB during fracture healing in chondrocytes of human enchondral tissue. Furthermore, BDNF is absent in mature bone. Taken together, BDNF and TrkB are involved in vessel formation and osteogenic processes during human fracture healing. The detection of BDNF and its TrkB receptor during various stages of the bone formation process in human fracture gap tissue were shown for the first time. The current study reveals that both proteins are up-regulated in human osteoblasts and endothelial cells in fracture healing.

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures.

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.

Light- and transmission-electron-microscopic investigations on distribution of CD44, connexin 43 and actin cytoskeleton during the foreign body reaction to a nanoparticular hydroxyapatite in mini-pigs.

Foreign body giant cells (FBGCs) are formed by fusion of mononucleated macrophages during the foreign body response to a nanoparticulate hydroxyapatite (HA) implanted in defects of mini-pig femura. The molecular mechanisms underlying the formation of FBGCs are still largely obscure. Here we propose connexin 43 (cx43) and CD44 as candidate molecules involved in the fusion process. Immunohistochemistry and ultrastructural immunogold labeling indicated that cx43 is present within the ruffled border of FBGCs and is the main component of gap junctions formed between fusing macrophages. CD44 was strongly expressed during clustering and fusion of mononucleated macrophages. FBGCs adhering apically at the implanted HA showed CD44 reactivity only along the basolateral aspects of the plasma membranes, while podosome formation was observed within the sealing zone and ruffled border. Taken together, these findings demonstrate that cx43 and CD44 are part of the fusion machinery responsible for the formation of FBGCs. Furthermore, the results of microfilament and cx43 labeling suggest a functional role for podosomes and hemi-channels in biomaterial degradation.

Quantitative analyses of bone composition in acetylcholine receptor M3R and alpha7 knockout mice.

AIMS: Increasing collagen synthesis was observed in lung after stimulation of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) on fibroblasts. Since collagen synthesis is an important process during fracture healing and bone remodelling, we asked whether cholinergic receptors are involved in bone collagen production. MAIN METHODS: In the present study we analysed 16 week old male knockout mice for nAChRα7 (α7-KO) and mAChR M3R (M3R-KO) in correlation to their corresponding wild types (WT). Microarchitecture of right femora, vertebrae Th13 and L1 were analysed by 3D Micro-CT, left femora by a three-point bending test and humeri by real-time RT-PCR. KEY FINDINGS: A significant decrease in relative bone volume, trabecular thickness, trabecular number, bone surface density, and a significant increase in trabecular separation and structure model index were measured for the M3R-KO using Micro-CT analysis. Bending stiffness of M3R-KO was significantly reduced in comparison to WT as well as the collagen 1α1 and 1α2 mRNA expression was down-regulated. No changes were detected for α7-KO using Micro-CT, biomechanical testing, and collagen mRNA expression. SIGNIFICANCE: Our results indicate that nAChRα7 are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis. Since the M3R-KO exhibit bone structures similar to systemically diseased bone it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities.

Elution kinetics, antimicrobial efficacy, and degradation and microvasculature of a new gentamicin-loaded collagen fleece.

Management of bone and soft tissue infections generally includes surgical procedures as well as attendant treatment and prevention with gentamicin-loaded fleeces. Conventional gentamicin-containing collagen fleeces currently in use are strongly acidic and exhibit limited biocompatibility thereby adversely affecting wound healing. To improve the antibiotic delivery system, a new phosphate-buffered, gentamicin-loaded fleece with pH-neutral properties has been developed (Jason G). This study aimed at comparing the elution kinetics of gentamicin release and the antimicrobial efficacy of conventional fleeces with the newly developed fleece in vitro. In addition, degradation and microvasculature of implanted fleeces were examined in a rat model and assessed using histology, as well as detection of ED-1 and PECAM-expression using immunohistochemistry. We show that the phosphate-buffered fleeces have reduced release (p < 0.05) of the integrated gentamicin. However, all of the fleeces tested had a significant antimicrobial effect on the growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains (p < 0.01). Among the fleeces tested, the new Jason G fleece had the weakest but nevertheless sufficient antimicrobial effectiveness. Evaluation of the antibiotic effect in the prevention of an infection showed no differences between the applied fleeces. Following surgical implantation of fleece in the backs of Wistar rats we observed, on day 5 after implantation, an increase in cell infiltration and microvascularization with the phosphate-buffered fleece as compared with conventional fleeces, which show necrotic cells on their surface. Unlike the acidic fleeces, on day 15 after implantation the pH-neutral fleece was resorbed widely. Here, we show that the new, pH-neutral, gentamicin-containing fleece Jason G exhibits good overall antimicrobial effectiveness against both gram-positive and gram-negative bacteria in vitro with improved degradation properties and microvasculature formation in vivo.

Enhancement of bone formation in hydroxyapatite implants by rhBMP-2 coating.

The combination of hydroxyapatite (HA) implants serving as osteoconductive scaffold with growth factors is an interesting approach for the improvement of bone defect healing. The purpose of this study was to test whether recombinant human bone morphogenetic protein-2 (rhBMP-2) coating of solid HA-implants improves bone formation in a cortical bone defect. Cylindrical trephine mill defects (diameter: 9.8 mm, depth: 10 mm) were created into the cortical tibia shaft of minipigs and subsequently filled either by plain HA cylinders (Endobon) or by rhBMP-2-coated HA cylinders. Fluorochrome labeling for the evaluation of time-dependent bone formation was done on days 8, 9, and 10 postsurgery with tetracyclin-100, at days 25 and 30 with alizarin-komplexon, and finally on days 32, 37, 73, and 79 with calcein green. Twelve weeks after implantation, the tibiae were harvested and were prepared for standard histological staining, fluorochrome analysis, and histomorphometry. Coating of HA implants with rhBMP-2 led to significant enhanced new bone formation of 84.7% (+/-4.6%) of the implant area with almost complete bony incorporation compared with only 27.7% (+/-8.5%) in the uncoated HA implants (p = 0.028). In both types of implants, osteoconduction of HA led to bone ingrowth of the surrounding host bone into the implants. However, only rhBMP-2-coated implants showed multitopic de novo bone formation reflecting the osteoinductive properties of rhBMP-2 in all areas of the HA implant. This study showed that the coating of HA ceramic implants with rhBMP-2 can significantly enhance new bone formation attributable to its osteoinductive effects.

Effects of platelet factors on biodegradation and osteogenesis in metaphyseal defects filled with nanoparticular hydroxyapatite--an experimental study in minipigs.

There are no studies on the cellular activity in the early phase of biodegradation and bone healing of bone substitutes loaded with platelet factors (PLF). The purpose of this study was to evaluate the cellular effects of PLF in combination with nanoparticulate hydroxyapatite (HA) on the biodegradation and bone formation after 20 days. Autogenous PLFs were obtained by centrifugation of miniature pig blood samples and subsequent degranulation of platelets by calcium and thrombin. A cylindrical bone defect with a diameter of 8.9 mm was created in the distal femoral condyle of 20 miniature pigs. Four of the defects were left empty, 8 were filled with HA with loading and 8 with HA loaded with PLF. The distal femur was harvested after 20 days and TRAP-staining, cathepsin-K and CD44 staining and scanning electron microscopy were performed for cellular assessment of biodegradation was done. Histomorphometry of new bone formation and of biodegradation of HA material was performed. PLF loading of HA led to statistically significant more TRAP-positive cells with enhanced biodegradation of the nanoparticulate HA but no statistically enhanced new bone formation compared to unloaded HA. Furthermore, there was a higher number of CD44 and cathepsin-K positive cells by PLF-loading. In summary, PLF led to stimulation of the cellular process of the biodegradation of HA.