Volker Alt

Delayed union and nonunions: epidemiology, clinical issues, and financial aspects.

Fracture healing is a critically important clinical event for fracture patients and for clinicians who take care of them. The clinical evaluation of fracture healing is based on both radiographic findings and clinical findings. Risk factors for delayed union and nonunion include patient dependent factors such as advanced age, medical comorbidities, smoking, non-steroidal anti-inflammatory use, various genetic disorders, metabolic disease and nutritional deficiency. Patient independent factors include fracture pattern, location, and displacement, severity of soft tissue injury, degree of bone loss, quality of surgical treatment and presence of infection. Established nonunions can be characterised in terms of biologic capacity, deformity, presence or absence of infection, and host status. Hypertrophic, oligotrophic and atrophic radiographic appearances allow the clinician to make inferences about the degree of fracture stability and the biologic viability of the fracture fragments while developing a treatment plan. Non-unions are difficult to treat and have a high financial impact. Indirect costs, such as productivity losses, are the key driver for the overall costs in fracture and non-union patients. Therefore, all strategies that help to reduce healing time with faster resumption of work and activities not only improve medical outcome for the patient, they also help reduce the financial burden in fracture and non-union patients.

Biocompatibility of silver nanoparticles and silver ions in primary human mesenchymal stem cells and osteoblasts

The prevention of implant-related infections is an important issue in medical research. The aim is to exploit the strong antimicrobial effect of silver nanoparticles (AgNP) to develop new antibacterial coatings for implants. However, there is still a serious lack of information on the influence of AgNP on bone metabolism. In the present study we have evaluated the influence of AgNP on cell stress, viability, proliferation and differentiation of primary human mesenchymal stem cells (MSC) and osteoblasts (OB). Finally, cellular uptake of the AgNP was examined. After 21 days impairment of cell viability of MSC and OB occurred at a concentration of 10 μg/g of AgNP. Cytotoxicity and inhibition of proliferation was highly time and dose dependent. No influence on cell differentiation, but an increase in cell stress, was observed. Uptake of AgNP into MSC and OB could be confirmed. In summary, these results demonstrate AgNP-mediated cytotoxicity at higher concentrations. Therefore, a therapeutical window for the application of AgNP in medical products might exist. However, the antibacterial benefits and potential health risks of AgNP need to be weighed in further studies.

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.

In Vitro Testing of Antimicrobial Activity of Bone Cement

ABSTRACT The purpose of this study was to establish a reliable and cost-effective microplate proliferation assay for in vitro antimicrobial testing of bone cement samples. Cement samples devoid of antimicrobial agents, loaded with 2% gentamicin or with different concentrations of high-porosity silver, were incubated in a 96-well microplate with several staphylococcal, Pseudomonas aeruginosa, and Enterococcus faecium isolates exhibiting different susceptibilities to gentamicin. After being rinsed, the samples were brought into a soy medium in which adherent cells on the cement surface either were killed by the antimicrobial surface or started to release clonal counterparts. The medium was monitored in real time by recording a time proliferation curve for each well. Microplate testing revealed no antibacterial effect of plain bone cement. The antibacterial activity of gentamicin-loaded bone cement was shown by the microplate test to depend on the gentamicin susceptibilities of the strains. The effect of high-porosity silver was dose dependent. Bactericidal activity against all tested strains was found for bone cement loaded with 1% high-porosity silver. The accuracy of this new proliferation assay was shown by the high correlation between the types of proliferation curves and antibiotic susceptibility. In contrast to routine agar diffusion testing, it assesses the dynamic response of microorganisms to antimicrobial agents in biomaterials and allows high-throughput screening and detection of antimicrobial properties of poorly water-soluble compounds like silver.

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 α3 and α5. RNA for CarAT, OCT-1, M2R, M5R, nAChR subunits α3, α5, α9, α10, β2, β3, 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 α7. Immunhistochemistry localized the nAChR subunits α3 and α5 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.

Calcium phosphate phases integrated in silica/collagen nanocomposite xerogels enhance the bioactivity and ultimately manipulate the osteoblast/osteoclast ratio in a human co-culture model

A human co-culture model of osteoblasts and osteoclasts, derived from bone marrow stromal cells and monocytes respectively, was used to characterize the influence of biomaterial modification on the bioactivity and ultimately the ratio of bone-forming to bone-resorbing cells cultivated directly on the surface. Nanocomposites of silica and collagen have been shown to function as skeletal structures in nature and were reproduced in vitro by using a sol-gel approach. The resulting xerogels exhibit a number of features that make it a valuable system for the development of innovative materials for bone substitution applications. In the present study, the incorporation of different calcium phosphate phases in silica/collagen-based gels was demonstrated to enhance the bioactivity of these samples. This ability of the biomaterial to precipitate calcium phosphate on the surface when incubated in simulated body fluids or cell culture medium is generally considered to an advantageous property for bone substitution materials. By co-cultivating human osteoblasts and osteoclasts up to 42 days on the xerogels, we demonstrate that the long-term ratio of these cell types depends on the level of bioactivity of the substrate samples. Biphasic silica/collagen xerogels exhibited comparably low bioactivity but encouraged proliferation of osteoblasts in comparison to osteoclast formation. A balanced ratio of both cell types was detected for moderately bioactive triphasic xerogels with 5% calcium phosphate. However, enhancing the bioactivity of the xerogel samples by increasing the calcium phosphate phase percentage to 20% resulted in a diminished number of osteoblasts in favor of osteoclast formation. Quantitative evaluation was carried out by biochemical methods (calcium, DNA, ALP, TRAP 5b) as well as RT-PCR (ALP, BSP II, OC, RANKL, TRAP, CALCR, VTNR, CTSK), and was supported by confocal laser scanning microscopy (cell nuclei, actin, CD68, TRAP) as well as scanning electron microscopy.

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.

Calcium Phosphate-Based Bone Substitutes

Background:The replacement of bone by means of foreign materials was already carried out in prehistoric times. Nowadays autogenous bone grafting is designated as the “golden standard” to fill large osseous defects which result from traumas, tumors, or birth defects. However, its disadvantages such as limited supply of autogenous bone and donor site morbidity have favored the use of bone substitutes. As these materials are characterized by their unlimited availability without bearing the risk of disease transmission, research on improving bone tissue healing by using bone substitutes of synthetic or biological origin is a field of major interest.Focus of Interest:Bone substitutes used clinically in orthopedics, periodontics, oral and maxillofacial surgery as well as in plastic, trauma, and reconstructive surgery comprise a wide variety of materials and have been the focus of interest for the last 80 years. The present review has focused on the frequently used calcium phosphate-based bone substitutes revealing either resorbable or nonresorbable properties. Their excellent biocompatibility due to their close mimicking of the inorganic phase of the natural bone mineral has led to their widespread use in bone reconstructive surgery.Examination Tools:Physicochemical properties of the materials have been shown by X-ray diffraction and scanning electron microscopy, whereas bioreactivity has been investigated by means of comparative histological evaluations and the use of various animal models. Transmission electron microscopy has been suitable for studying cell-mediated degradation at the cellular level. The results are discussed with special regard to the origin, composition, and general characteristics of inorganic bone substitutes.

Effects of gentamicin and gentamicin–RGD coatings on bone ingrowth and biocompatibility of cementless joint prostheses: An experimental study in rabbits

Antimicrobial coatings are of interest as a means to improve infection prophylaxis in cementless joint arthroplasty. However, those coatings must not interfere with the essential bony integration of the implants. Gentamicin-hydroxyapatite (gentamicin-HA) and gentamicin-RGD (arginine-glycine-aspartate)-HA coatings have recently been shown to significantly reduce infection rates in a rabbit infection prophylaxis model. The purpose of the current study was to investigate the in vitro elution kinetics and in vivo effects of gentamicin-HA and gentamicin-RGD-HA coatings on new bone formation, implant integration and biocompatibility in a rabbit model. In vitro elution testing showed that 95% and 99% of the gentamicin was released after 12 and 24 h, respectively. The in vivo study comprised 45 rabbits in total, with six animals for each of the gentamicin-HA, gentamicin-RGD-HA group and control pure HA coating groups for the 4 week time period, and nine animals for each of the three groups for the 12 week observation period. A 2.0 mm steel K-wire with one of the coatings under test was placed in the intramedullary canal of the tibia. After 4 and 12 weeks the tibiae were harvested and three different areas (proximal metaphysis, shaft area, distal metaphysis) were assessed by quantitative and qualitative histology for new bone formation, direct implant-bone contact and the formation of multinucleated giant cells. The results exhibited high standard deviations in all subgroups. There was a trend towards better bone formation and better direct implant contact in the pure HA coating group compared with both gentamicin coatings after 4 and 12 weeks, which was, however, not statistically significant. The number of multinucleated giant cells did not differ significantly between the three groups at both time points. In summary, both gentamicin coatings with 99% release of gentamicin within 24 h revealed good biocompatibility and bony integration, which was not statistically significant different compared with pure HA coating. Limitations of the study are the high standard deviation of the results and the limited number of animals per time point.

Induction of osteoporosis with its influence on osteoporotic determinants and their interrelationships in rats by DEXA

Summary Background As women are the population most affected by multifactorial osteoporosis, research is focused on unraveling the underlying mechanism of osteoporosis induction in rats by combining ovariectomy (OVX) either with calcium, phosphorus, vitamin C and vitamin D2/D3 deficiency, or by administration of glucocorticoid (dexamethasone). Material/Methods Different skeletal sites of sham, OVX-Diet and OVX-Steroid rats were analyzed by Dual Energy X-ray Absorptiometry (DEXA) at varied time points of 0, 4 and 12 weeks to determine and compare the osteoporotic factors such as bone mineral density (BMD), bone mineral content (BMC), area, body weight and percent fat among different groups and time points. Comparative analysis and interrelationships among osteoporotic determinants by regression analysis were also determined. Results T scores were below-2.5 in OVX-Diet rats at 4 and 12 weeks post-OVX. OVX-diet rats revealed pronounced osteoporotic status with reduced BMD and BMC than the steroid counterparts, with the spine and pelvis as the most affected skeletal sites. Increase in percent fat was observed irrespective of the osteoporosis inducers applied. Comparative analysis and interrelationships between osteoporotic determinants that are rarely studied in animals indicate the necessity to analyze BMC and area along with BMD in obtaining meaningful information leading to proper prediction of probability of osteoporotic fractures. Conclusions Enhanced osteoporotic effect observed in OVX-Diet rats indicates that estrogen dysregulation combined with diet treatment induces and enhances osteoporosis with time when compared to the steroid group. Comparative and regression analysis indicates the need to determine BMC along with BMD and area in osteoporotic determination.