Composite material consisting of microporous beta-TCP ceramic and alginate-dialdehyde-gelatin for controlled dual release of clindamycin and bone morphogenetic protein 2.

The aim of this study was to produce a composite of microporous ß-TCP filled with alginate-gelatin crosslinked hydrogel, clindamycin and bone morphogenetic protein (BMP-2) to prolong the drug-release behaviour for up to 28 days. The most promising alginate-di-aldehyde(ADA)-gelatin gel for drug release from microcapsules was used to fill microporous ß-TCP ceramics under directional flow in a special loading chamber. Dual release of clindamycin and BMP-2 was measured on days 1, 2, 3, 6, 9, 14, 21 and 28 by high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). After release, the microbial efficacy of the clindamycin was checked and the biocompatibility of the composite was tested in cell culture. Clindamycin and the model substance FITC-protein A were released from microcapsules over 28 days. The clindamycin burst release was 43 ± 1%. For the loaded ceramics, a clindamycin release above the minimal inhibitory concentration (MIC) until day 9 and a burst release of 90.56 ± 2.96% were detected. BMP-2 was released from the loaded ceramics in low concentrations over 28 days. The release of active substances from ß-TCP and hydrogel have already been extensively studied. Directional flow loading is a special procedure in which the ceramic could act as a stabilizer in the bone and, as a biodegradable system, enables a single-stage surgical procedure. Whether ADA-gelatin gel is suitable for this procedure as a more biodegradable alternative to pure alginate or whether a dual release is possible in this composite has not yet been investigated.

A human bone infection organ model for biomaterial research.

The aim of this work was to establish an organ model for staphylococcal infection of human bone samples and to investigate the influence and efficacy of a microporous ß-tricalcium phosphate ceramic (ß-TCP, RMS Foundation) loaded with hydrogels (alginate, alginate-di-aldehyde (ADA)-gelatin) and clindamycin on infected human bone tissue over a period of 28 days. For this purpose, human tibia plateaus, collected during total knee replacement surgery, were used as a source of bone material. Samples were infected with S. aureus ATCC29213 and treated with differently loaded ß-TCP composites (alginate +/- clindamycin, ADA-gelatin +/- clindamycin, unloaded). The loading of the composites was carried out by means of a flow chamber. The infection was observed for 28 days, quantifying bacteria in the medium and the osseus material on day 1, 7, 14, 21 and 28. All samples were histologically processed for bone vitality evaluation. Bone infection could be consistently performed within the organ model. In addition, a strong reduction in bacterial counts was recorded in the groups treated with ADA-gelatin + clindamycin and alginate + clindamycin, while the bacterial count in the control groups remained constant. No significant differences between groups could be observed in the number of lacunae filled with osteocytes suggesting no differences in bone vitality among groups. In an ex-vivo human bone infection model, over a period of 28 days bacterial growth could be reduced by treatment with ADA-Gel + CLI and ALG + CLI -releasing ß-TCP composites. This could be relevant for its clinical use. Further work will be necessary to improve the loading of ß-TCP and the bone infection organ model itself. STATEMENT OF SIGNIFICANCE: The common treatment of bone infections is debridement and systemic administration of antibiotics. In some cases, antibiotic-containing carriers are already used, but these must be removed again. Our work is intended to show another treatment option. The scaffold we have developed, made of a calcium phosphate ceramic and a hydrogel as the active substance carrier, can, in addition to releasing the active substance, also assume a load-bearing function of the bone and is biodegradable. In addition, the model we developed can also be used for the analysis and treatment of bone infections other than those of the musculoskeletal system. More importantly, it can also serve as a substitute for previously used animal experiments.

A Method for the Evaluation of Early Osseointegration of Implant Materials Ex Vivo: Human Bone Organ Model.

In the present work, an ex vivo organ model using human bone (explant) was developed for the evaluation of the initial osseointegration behavior of implant materials. The model was tested with additive manufactured Ti6Al4V test substrates with different 3D geometries. Explants were obtained from patients who underwent total knee replacement surgery. The tibial plateaus were used within 24 h after surgery to harvest bone cylinders (BC) from the anterior side using hollow burrs. The BCs were brought into contact with the test substrate and inserted into an agarose mold, then covered with cell culture media and subjected to the external load of 500 g. Incubation was performed for 28 days. After 28d the test substrate was removed for further analysis. Cells grown out BC onto substrate were immunostained with DAPI and with an antibody against Collagen-I and alkaline phosphatase (ALP) for visualization and cell counting. We show that cells stayed alive for up to 28d in our organ model. The geometry of test substrates influences the number of cells grown onto substrate from BCs. The model presented here can be used for testing implant materials as an alternative for in vitro tests and animal models.

The future of basic science in orthopaedics and traumatology: Cassandra or Prometheus?

Orthopaedic and trauma research is a gateway to better health and mobility, reflecting the ever-increasing and complex burden of musculoskeletal diseases and injuries in Germany, Europe and worldwide. Basic science in orthopaedics and traumatology addresses the complete organism down to the molecule among an entire life of musculoskeletal mobility. Reflecting the complex and intertwined underlying mechanisms, cooperative research in this field has discovered important mechanisms on the molecular, cellular and organ levels, which subsequently led to innovative diagnostic and therapeutic strategies that reduced individual suffering as well as the burden on the society. However, research efforts are considerably threatened by economical pressures on clinicians and scientists, growing obstacles for urgently needed translational animal research, and insufficient funding. Although sophisticated science is feasible and realized in ever more individual research groups, a main goal of the multidisciplinary members of the Basic Science Section of the German Society for Orthopaedics and Trauma Surgery is to generate overarching structures and networks to answer to the growing clinical needs. The future of basic science in orthopaedics and traumatology can only be managed by an even more intensified exchange between basic scientists and clinicians while fuelling enthusiasm of talented junior scientists and clinicians. Prioritized future projects will master a broad range of opportunities from artificial intelligence, gene- and nano-technologies to large-scale, multi-centre clinical studies. Like Prometheus in the ancient Greek myth, transferring the elucidating knowledge from basic science to the real (clinical) world will reduce the individual suffering from orthopaedic diseases and trauma as well as their socio-economic impact.

Development of Bioinspired Functional Chitosan/Cellulose Nanofiber 3D Hydrogel Constructs by 3D Printing for Application in the Engineering of Mechanically Demanding Tissues.

Soft tissues are commonly fiber-reinforced hydrogel composite structures, distinguishable from hard tissues by their low mineral and high water content. In this work, we proposed the development of 3D printed hydrogel constructs of the biopolymers chitosan (CHI) and cellulose nanofibers (CNFs), both without any chemical modification, which processing did not incorporate any chemical crosslinking. The unique mechanical properties of native cellulose nanofibers offer new strategies for the design of environmentally friendly high mechanical performance composites. In the here proposed 3D printed bioinspired CNF-filled CHI hydrogel biomaterials, the chitosan serves as a biocompatible matrix promoting cell growth with balanced hydrophilic properties, while the CNFs provide mechanical reinforcement to the CHI-based hydrogel. By means of extrusion-based printing (EBB), the design and development of 3D functional hydrogel scaffolds was achieved by using low concentrations of chitosan (2.0-3.0% (w/v)) and cellulose nanofibers (0.2-0.4% (w/v)). CHI/CNF printed hydrogels with good mechanical performance (Young's modulus 3.0 MPa, stress at break 1.5 MPa, and strain at break 75%), anisotropic microstructure and suitable biological response, were achieved. The CHI/CNF composition and processing parameters were optimized in terms of 3D printability, resolution, and quality of the constructs (microstructure and mechanical properties), resulting in good cell viability. This work allows expanding the library of the so far used biopolymer compositions for 3D printing of mechanically performant hydrogel constructs, purely based in the natural polymers chitosan and cellulose, offering new perspectives in the engineering of mechanically demanding hydrogel tissues like intervertebral disc (IVD), cartilage, meniscus, among others.

Mechanical Properties of the Composite Material consisting of ß-TCP and Alginate-Di-Aldehyde-Gelatin Hydrogel and Its Degradation Behavior.

This work aimed to determine the influence of two hydrogels (alginate, alginate-di-aldehyde (ADA)/gelatin) on the mechanical strength of microporous ceramics, which have been loaded with these hydrogels. For this purpose, the compressive strength was determined using a Zwick Z005 universal testing machine. In addition, the degradation behavior according to ISO EN 10993-14 in TRIS buffer pH 5.0 and pH 7.4 over 60 days was determined, and its effects on the compressive strength were investigated. The loading was carried out by means of a flow-chamber. The weight of the samples (manufacturer: Robert Mathys Foundation (RMS) and Curasan) in TRIS solutions pH 5 and pH 7 increased within 4 h (mean 48 ± 32 mg) and then remained constant over the experimental period of 60 days. The determination surface roughness showed a decrease in the value for the ceramics incubated in TRIS compared to the untreated ceramics. In addition, an increase in protein concentration in solution was determined for ADA gelatin-loaded ceramics. The macroporous Curasan ceramic exhibited a maximum failure load of 29 ± 9.0 N, whereas the value for the microporous RMS ceramic was 931 ± 223 N. Filling the RMS ceramic with ADA gelatin increased the maximum failure load to 1114 ± 300 N. The Curasan ceramics were too fragile for loading. The maximum failure load decreased for the RMS ceramics to 686.55 ± 170 N by incubation in TRIS pH 7.4 and 651 ± 287 N at pH 5.0.

Inverse 3D Printing with Variations of the Strand Width of the Resulting Scaffolds for Bone Replacement.

The objective of this study was to vary the wall thicknesses and pore sizes of inversely printed 3D molded bodies. Wall thicknesses were varied from 1500 to 2000 to 2500 µm. The pores had sizes of 500, 750 and 1000 µm. The sacrificial structures were fabricated from polylactide (PLA) using fused deposition modeling (FDM). To obtain the final bioceramic scaffolds, a water-based slurry was filled into the PLA molds. The PLA sacrificial molds were burned out at approximately 450 °C for 4 h. Subsequently, the samples were sintered at 1250 °C for at least 4 h. The scaffolds were mechanically characterized (native and after incubation in simulated body fluid (SBF) for 28 days). In addition, the biocompatibility was assessed by live/dead staining. The scaffolds with a strand spacing of 500 µm showed the highest compressive strength; there was no significant difference in compressive strength regardless of pore size. The specimens with 1000 µm pore size showed a significant dependence on strand width. Thus, the specimens (1000 µm pores) with 2500 µm wall thickness showed the highest compressive strength of 5.97 + 0.89 MPa. While the 1000(1500) showed a value of 2.90 + 0.67 MPa and the 1000(2000) of 3.49 + 1.16 MPa. As expected for beta-Tricalciumphosphate (ß-TCP), very good biocompatibility was observed with increasing cell numbers over the experimental period.

Anteversion Angle Measurement in Suspected Torsional Malalignment of the Femur in 3-Dimensional EOS vs Computed Tomography-A Validation Study.

BACKGROUND: Computed tomography (CT) scan is the standard for assessment of femoral torsion. This observational study was conducted to evaluate the comparability of the EOS radiation dose scanning system (EOS imaging, Paris, France) and the CT scan in patients with suspected torsional malalignment of the femur. METHODS: Patients with suspected torsional malalignment of the femur were included in a study for surgical planning. The primary endpoint was to compare the 3-dimensional radiological (EOS) imaging system with the CT scan to determine femoral anteversion (AV) angle. Three independent raters performed measurements. Comparability of CT scan and EOS values was assessed by Pearson correlation, t test, interobserver reliability, and intraobserver reliability (Cronbach alpha). RESULTS: About 34 femora were examined. Interobserver reliability/intraobserver reliability was 0.911 of 0.955 for EOS and 0.934 of 0.934 for CT scan. EOS system revealed an AV angle of 12.2° ± 10.0° (-15.0° to 32.0°). CT examinations showed an AV angle of 12.6° ± 9.2° (-3.2° to 35.6°). About 11 hips featured physiological AV, 14 hips showed decreased AV (<10°) or retroversion (<0°), and 9 hips showed increased AV (>20°). Overall, a strong Pearson correlation of τ = 0.855 and a highly significant correlation in the t test for both methods was seen. In patients with decreased AV, retroversion, or increased AV, Pearson correlation only resulted in a moderate/low correlation of τ = 0.495 and τ = 0.292. The t test showed no significant correlation at malrotation. CONCLUSION: In torsional malalignment, EOS does not have correlation with CT measurements. In contrast to CT scan, EOS allows femoral torsion measurement independent of legs' positioning.

Histomorphological Alterations of Human Anterior Cruciate Ligament Grafts During Mid-Term and Long-Term Remodeling.

OBJECTIVE: The aim of the present paper is to analyze mid-term and long-term alterations of human anterior cruciate ligament (ACL) grafts during the remodeling process with special regards to cellularity, α-smooth muscle protein (αSMP) expression, and crimp length in comparison to the native ACL. METHODS: A total of 34 patients were included (23 male and 11 female). Biopsies of 13 semitendinosus tendon and 14 patellar tendon autografts were obtained during surgical revision secondary to an ACL reconstruction. According to the interval between the index procedure and sample collection, the patients were divided into four groups: 4-12 months, 13-60 months, 61-108 months, and >108 months. Seven samples of native ruptured ACL tissue obtained during surgical intervention served as control. All biopsies were taken from the intraligamentous part of the ACL or the graft. Histomorphological and immunohistochemical analyses were conducted after samples were stained using hematoxylin-eosin, Giemsa, and αSMP enzyme-labeled antibodies. The total cell density, the numbers of fibroblasts and fibrocytes, the fibroblast/fibrocyte ratio, the number of αSMP+ cell nuclei, and the percentage of αSMP+ cells per fibroblast as well as the crimp lengths were determined using light microscopy. RESULTS: In the early phase of remodeling, the grafts featured extensively high total cell counts (1021.2 ± 327.8, P = 0.001), with high numbers of fibroblasts (841.4 ± 245.2, P = 0.002), fibrocytes (174.5 ± 113.0, P = 0.04), and αSMP+ cells (78.3 ± 95.0, P = 0.02) compared to controls (390.1 ± 141.7, 304.5 ± 160.8, 65.6 ± 31.4 and 2.3 ± 2.6, respectively). Thereafter, the numbers of all cell entities decreased. After more than 108 months, the percentage of αSMP+ cells per fibroblast reached physiological values (ratio 1.3 ± 1.0, P = 0.41; control 0.8 ± 0.8), while the total cell count (834.3 ± 183.7, P = 0.001) as well as the numbers of fibroblasts (663.5 ± 192.6, P = 0.006) and fibrocytes (134.1 ± 73.0, P = 0.049) remained significantly high. The fibroblast/fibrocyte ratio showed no significant alterations over the course of time compared to the controls. The collagen crimp lengths were elongated by tendency in the early phase (28.8 ± 12.9 mm, P = 0.15; control 20.7 ± 2.2 mm) and significantly shortened over time, with the lowest values in the long term (14.8 ± 2.0 mm, P = 0.001). The comparison of biopsies from semitendinosus tendon and patellar tendon autografts revealed no significant differences for any of the histomorphological parameters investigated. CONCLUSION: This study reveals distinctive mid-term and long-term immunomorphological alterations during human ACL graft remodeling. These data clearly indicate that the remodeling is a process that continues for 9 years or more. Furthermore, it seems to be a process of adaptation rather than full restoration. Even in the long run, several biological properties of the native ACL are not completely reestablished.

The acromioclavicular ligament shows an early and dynamic healing response following acute traumatic rupture.

PURPOSE: Symptomatic horizontal instability is clinically relevant following acute acromioclavicular joint dislocations. However, the intrinsic healing response is poorly understood. The present study sought to investigate time-dependent healing responses of the human acromioclavicular ligament following acute traumatic rupture. METHODS: Biopsies of the acromioclavicular ligament were obtained from patients undergoing surgical treatment for acute acromioclavicular joint dislocations. Specimens were stratified by time between trauma and surgery: group 1, 0-7 days (n = 5); group 2, 8-14 days (n = 6); and group 3, 15-21 days (n = 4). Time-dependent changes in cellularity, collagen (type 1 and 3) concentration, and histomorphological appearance were evaluated for the rupture and intact zone of the acromioclavicular ligament. RESULTS: Group 1 was characterized by cellular activation and early inflammatory response. The rupture zone exhibited a significantly higher count of CD68-positive cells than the intact zone (15.2 vs 7.4; P ≤ 0.05). Consistently, synovialization of the rupture end was observed. Within the second week, the rupture zone was subject to proliferation showing more fibroblast-like cells than the intact zone (66.8 vs 43.8; P ≤ 0.05) and a peak of collagen type 3 expression (group 1: 2.2 ± 0.38, group 2: 3.2 ± 0.18, group 3: 2.8 ± 0.57; P ≤ 0.05). Signs of consolidation and early remodeling were seen in the third week. CONCLUSIONS: The acromioclavicular ligament exhibits early and dynamic healing responses following acute traumatic rupture. Our histological findings suggest that surgical treatment of acute ACJ dislocations should be performed as early as possible within a timeframe of 1 week after trauma to exploit the utmost biological healing potential. Prospective clinical studies are warranted to investigate whether early surgical treatment of ACJ dislocations translates into clinical benefits.

Acute Joint Blockage due to Abrasion-Related Dislocation of a Silastic Radial Head Prosthesis: A Histological Examination after 14 Years of Durability.

The implantation of a radial head prosthesis can take place as a therapeutic option after radial head fracture. There are various implants for this purpose. Many studies and case reports about silastic radial head prosthesis implantation describe foreign body reactions with accompanying synovitis and poor functional results. A few studies have investigated the reason for the material failure and the accompanying synovitis. The case report presented shows an unusually long durability of an in situ 14-year silastic radial head prosthesis. 14 years after implantation, a previously full-time working and healthy patient presented himself with a dislocation of the silastic radial head prosthesis and atraumatic joint blockage of the right elbow triggered by a negligible movement. The prosthesis was removed surgically. We found a macroscopic foreign body reaction intraoperatively. In a histopathological examination, with hematoxylin and eosin staining (HE) in 40x and 100x magnification, we have seen an aseptic inflammatory response to foreign bodies with activated epithelial cells and multinucleated giant cells with intracytoplasmic foreign material. Due to these problems, the silastic radial head prosthesis is no longer used today. However, there are still patients with the implanted silastic radial head prosthesis, which should therefore be checked regularly. A metal prosthesis also does not seem to be an optimal alternative due to cartilage wear and loss of ROM. The choice of prosthesis material should be selected carefully and patient-specific in radial head prosthetics according of the results presented.

Nanomechanical 3D Depth Profiling of Collagen Fibrils in Native Tendon.

Connective tissue displays a large compositional and structural complexity that involves multiple length scales. In particular, on the molecular and the nanometer level, the elementary processes that determine the biomechanics of collagen fibrils in connective tissues are still poorly understood. Here, we use atomic force microscopy (AFM) to determine the three-dimensional (3D) depth profiles of the local nanomechanical properties of collagen fibrils and their embedding interfibrillar matrix in native (unfixed), hydrated Achilles tendon of sheep and chickens. AFM imaging in air with controlled humidity preserves the tissue's water content, allowing the assembly of collagen fibrils to be imaged in high resolution beneath an approximately 5-10 nm thick layer of the fluid components of the interfibrillar matrix. We collect pointwise force-distance (FD) data and amplitude-phase-distance (APD) data, from which we construct 3D depth profiles of the local tip-sample interaction forces. The 3D images reveal the nanomechanical morphology of unfixed, hydrated collagen fibrils in native tendon with a 0.1 nm depth resolution and a 10 nm lateral resolution. We observe a diversity in the nanomechanical properties among individual collagen fibrils in their adhesive and in their repulsive, viscoelastic mechanical response as well as among the contact points between adjacent collagen fibrils. This sheds new light on the role of interfibrillar bonds and the mechanical properties of the interfibrillar matrix in the biomechanics of tendon.

Expression of xylosyltransferases I and II and their role in the pathogenesis of arthrofibrosis.

BACKGROUND: Arthrofibrosis is a painful and restraining complication that occurs after about 10% of total knee arthroplasty and cruciate ligament surgery. The pathogenesis of arthrofibrosis has not yet been fully understood. Stress signals stimulate immune cells, and fibroblast differentiates into myofibroblast, which produce a large amount of collagen. Xylosyltransferases also appear to be involved in these pathways. They catalyze proteoglycan biosynthesis, which is involved in tissue remodeling and myofibroblast differentiation. The aim of this study was to investigate the relationship between the disease arthrofibrosis and the expression of the two isoforms of xylosyltransferases I and II. METHODS: Tissue samples from 14 patients with arthrofibrosis were compared with tissue samples from seven healthy controls. The xylosyltransferases were detected by immunohistochemistry. The tissues were divided into four different areas of interest: vessels, synovialis, cell-poor and cell-rich fibrosis, or cell-poor and cell-rich areas in the control group. A quantification of the results was performed by modification of the immunoreactive score according to Remmele and Stegner. RESULTS: Xylosyltransferase I was expressed in the various tissue types at varying rates. Xylosyltransferase I expression was considerably and significantly stronger than that of xylosyltransferase II. The following sequences of xylosyltransferase I and xylosyltransferase II expression were determined as follows: vessels >> cell-rich fibrosis > cell-poor fibrosis > synovialis. A positive correlation between the number of positive fibroblasts and the immunoreactive scoring system (IRS) was documented. CONCLUSIONS: The significant positive correlation of xylosyltransferase -I expression with increasing number of fibroblasts demonstrates a high myofibroblast differentiation rate, which implies a gradual event as the pathogenesis of arthrofibrosis.

Biomechanical dynamic comparison of biodegradable pins and titanium screws for operative stabilization of displaced radial head fractures.

For radial head osteosynthesis, biodegradable implants are gaining in importance to minimize cartilage destruction and implant impingement and to supersede implant removal. Since loss of reduction and pseudarthrosis remain challenging complications, new implants should at least provide comparable biomechanical properties as commonly used metal implants. The objective of this study was to compare the treatment by polylactide pins to titanium screws and to quantify the produced cartilage defects. Eight pairs of human cadaver radii with a standardized Mason type II fracture were stabilized either by two 2.0-mm polylactide pins or titanium screws. The produced cartilage defects were quantified using an image analyzing software. Quasi-static loading was performed axially and transversally for 10 cycles each between 10 and 50 N. Afterward, implant loosening was tested by axial loading up to 10,000 cycles, followed by load to failure testing. Polylactide pins showed less construct stiffness under axial (p = 0.017) and transversal (p = 0.012) loading, and one polylactide pins construct failed after two cycles of transversal loading. At axial loading, a high correlation between bone mineral density and construct stiffness was observed among polylactide pins (R = 0.667; p = 0.071), which was not seen among titanium screws (R = 0.262; p = 0.531). No difference in implant loosening was recorded after 10,000 cycles (p = 0.237); however, one polylactide pins construct failed after 30 cycles and failure loads were higher for titanium screws (p=0.017). Polylactide pin produced smaller cartilage defects (p=0.012). In conclusion, simple radial head fractures treated by polylactide pins show less biomechanical stability than treated by titanium screws, particularly in osteoporotic bone which might lead to secondary loss of reduction. Due to smaller cartilage defects and equal properties under continuous loading, polylactide pins might represent a gentle alternative in patients with good bone quality making subsequent implant removal redundant.

Thin Degradable Coatings for Optimization of Osteointegration Associated with Simultaneous Infection Prophylaxis.

One of the most common causes of implant failure is aseptic prosthesis loosening. Another frequent complication after prosthesis implant is the microbial colonization of the prosthesis surface, which often leads to a replacement of the prosthesis. One approach to reduce these complications is the application of bioactive substances to implant surfaces. Both an antibiotic prophylaxis and a faster osteointegration can be obtained by incorporation of bactericidal active metals in degradable calcium phosphate (CaP) coatings. In this study, thin degradable calcium phosphate ceramic coatings doped with silver (Ag), copper (Cu), and bismuth (Bi) on a titanium substrate were prepared with the aid of the high-velocity suspension flame spraying (HVSFS) coating process. To characterize the samples surface roughness, brightfield microscopy of the coatings, X-ray diffraction (XRD)-analysis for definition of the phase composition of the layers, Raman spectroscopy for determination of the phase composition of the contained metals, element-mapping for Cu-content verification, release kinetics for detection of metal ions and ceramic components of the coatings were carried out. The aim of this study was to evaluate in vitro biocompatibility and antimicrobial activity of the coatings. For biocompatibility testing, growth experiments were performed using the cell culture line MG-63. Cell viability was investigated by Giemsa staining and live/dead assay. The WST-1 kit was used to quantify cell proliferation and vitality in vitro and the lactate dehydrogenase (LDH) kit to quantify cytotoxicity. The formation of hydroxyapatite crystals in simulated body fluid was investigated to predict bioactivity in vivo. The Safe Airborne Antibacterial Assay with Staphylococcus aureus (S. aureus) was used for antimicrobial testing. The results showed good biocompatibility of all the metal doped CaP coatings, furthermore Cu and Ag doped layers showed significant antibacterial effects against S. aureus.

Inversely 3D-Printed ß-TCP Scaffolds for Bone Replacement.

The aim of this study was to predefine the pore structure of ß-tricalcium phosphate (ß-TCP) scaffolds with different macro pore sizes (500, 750, and 1000 µm), to characterize ß-TCP scaffolds, and to investigate the growth behavior of cells within these scaffolds. The lead structures for directional bone growth (sacrificial structures) were produced from polylactide (PLA) using the fused deposition modeling techniques. The molds were then filled with ß-TCP slurry and sintered at 1250 °C, whereby the lead structures (voids) were burnt out. The scaffolds were mechanically characterized (native and after incubation in simulated body fluid (SBF) for 28 d). In addition, biocompatibility was investigated by live/dead, cell proliferation and lactate dehydrogenase assays. The scaffolds with a strand spacing of 500 µm showed the highest compressive strength, both untreated (3.4 ± 0.2 MPa) and treated with simulated body fluid (2.8 ± 0.2 MPa). The simulated body fluid reduced the stability of the samples to 82% (500), 62% (750) and 56% (1000). The strand spacing and the powder properties of the samples were decisive factors for stability. The fact that ß-TCP is a biocompatible material is confirmed by the experiments. No lactate dehydrogenase activity of the cells was measured, which means that no cytotoxicity of the material could be detected. In addition, the proliferation rate of all three sizes increased steadily over the test days until saturation. The cells were largely adhered to or within the scaffolds and did not migrate through the scaffolds to the bottom of the cell culture plate. The cells showed increased growth, not only on the outer surface (e.g., 500: 36 ± 33 vital cells/mm² after three days, 180 ± 33 cells/mm² after seven days, and 308 ± 69 cells/mm² after 10 days), but also on the inner surface of the samples (e.g., 750: 49 ± 17 vital cells/mm² after three days, 200 ± 84 cells/mm² after seven days, and 218 ± 99 living cells/mm² after 10 days). This means that the inverse 3D printing method is very suitable for the presetting of the pore structure and for the ingrowth of the cells. The experiments on which this work is based have shown that the fused deposition modeling process with subsequent slip casting and sintering is well suited for the production of scaffolds for bone replacement.

Viscoelasticity and histology of the human cartilage in healthy and degenerated conditions of the knee.

BACKGROUND: There are many studies on osteoarthritis, but only a few studies deal with human arthrosis, comparing the mechanical properties of healthy and diseased samples. In most of these studies, only isolated areas of the tibia are examined. There is currently only one study investigating the complete mapping of cartilage tissue but not the difference between instantaneous modulus (IM) in healthy and diseased samples. The aim of this study is to investigate the relationship between the biomechanical and histological changes of articular cartilage in the pathogenesis of osteoarthritis. METHODS: The study compared 25 tibiae with medial gonarthrosis and 13 healthy controls. The IM was determined by automated indentation mapping using a Mach-1 V500css testing machine. A grid was projected over the sample and stored so that all measurements could be taken at the same positions (100 ± 29 positions across the tibiae). This grid was then used to perform the thickness measurement using the needle method. Samples were then taken for histological examinations using a hollow milling machine. Then Giemsa and Safranin O staining were performed. In order to determine the degree of arthrosis according to histological criteria, the assessment was made with regard to Osteoarthritis Research Society International (OARSI) and AHO scores. RESULTS: A significant difference (p < 0.05) could be observed in the measured IM between the controls with 3.43 ± 0.36 MPa and the samples with 2.09 ± 0.18 MPa. In addition, there was a significant difference in IM in terms of meniscus-covered and meniscus-uncovered areas. The difference in cartilage thickness between 2.25 ± 0.11 mm controls and 2.0 ± 0.07 mm samples was highly significant with p < 0.001. With regard to the OARSI and AHO scores, the samples differed significantly from the controls. The OARSI and AHO scores showed a significant difference between meniscus-covered and meniscus-uncovered areas. CONCLUSIONS: The controls showed significantly better viscoelastic behavior than the arthrotic samples in the measured IM. The measured biomechanical values showed a direct correlation between histological changes and altered biomechanics in gonarthrosis.

Alendronate Release from UHMWPE-Based Biomaterials in Relation to Particle Size of the GUR Powder for Manufacturing.

Ultra-high molecular weight polyethylene (UHMWPE) is widely used in endoprosthetics and has been the subject of countless studies. This project investigates the dependence of alendronate (AL) release on the molecular weight of the UHMWPE used (GUR1020 and GUR1050). A 0.5 wt% AL was added to the UHMWPE during the production of the moldings. In addition to the 14-day release tests, biocompatibility tests such as live dead assay, cell proliferation assay (WST) and Lactate dehydrogenase test (LDH) with MG-63 cells as well as a tensile test according to DIN EN ISO 527 were carried out. The released AL concentration was determined by HPLC. A continuous release of the AL was observed over the entire period of 2 weeks. In addition, a correlation between molar mass and AL release was demonstrated. The GUR1020 showed a release four times higher than the GUR1050. Both materials have no negative influence on the proliferation of MG-63 cells. This was also confirmed in the live/dead assay by the increase in cell count. No cytotoxicity was detected in the LDH test. The addition of 0.5 wt% AL increased the elongation at break for GUR1020 by 23% and for GUR1050 by 49%. It was demonstrated that the choice of UHMWPE has an influence on the release of AL. The particle size in particular has a strong influence on the release behavior.

Immunohistochemical examination in arthrofibrosis of the knee joint.

INTRODUCTION: Arthrofibrosis (AF) is the result of increased cell proliferation and synthesis of matrix proteins (collagen I, III, and VI). Especially after invasive knee surgery, e.g., ligament reconstruction or knee replacement, abnormal fibroblast proliferation with pathological periarticular fibrosis can be observed leading to severely limited joint motion. The pathogenesis of AF is currently not fully understood. The present work aims to determine pathogenic factors. MATERIALS AND METHODS: A descriptive, histological and immunohistochemical comparative study was performed on tissue samples of 14 consecutive patients undergoing arthrolysis for joint stiffness due to AF. Seven human autopsy specimens served as control. Samples were stained for expression of relevant markers such as CD68, α-smooth muscle actin (ASMA), beta-catenin, BMP-2 and examined for the histological grade of AF (cell-rich versus cell-poor) and compared to a control. Furthermore, a microscopic evaluation of the samples for cell differentiation and number was performed. RESULTS: Tissue sections of cell-rich fibrosis showed a significantly higher expression of CD68 compared to the control with less than 10% of CD68 positive cells (p = 0.002). In cell-poor fibrosis no statistically significant difference was obvious (p = 0.228). Expression of ASMA in synovia, vessels, cell-rich and cell-poor fibrosis showed median values of 2.00 in the AF group and 1.75 in the control. Both groups differed significantly (p = 0.003). AF tissue showed a significantly difference in expression of ß-catenin (p < 0.001) compared to the control. The overall difference between AF and control group in expression of BMP-2 was also statistically significant (p = 0.002). CONCLUSIONS: Expression of CD68, ASMA, beta-catenin and BMP-2 is significantly increased in AF tissue samples. Based on presented findings, histological evaluation and immunohistochemical assessment of CD68, ASMA, ß-catenin and BMP-2 expression may proof useful to diagnose AF and to analyze AF activity.

Single-Bundle Versus Double-Bundle Anterior Cruciate Ligament Reconstruction-5-Year Results.

PURPOSE: To compare anatomic single-bundle (SB) with double-bundle (DB) anterior cruciate ligament reconstruction (ACL-R) and to evaluate the respective clinical outcome results. METHODS: In a prospective randomized study, 64 patients were included and separated into 2 groups. Anatomic SB and DB ACL-Rs were performed with hamstring tendons. Five years after surgery, the follow-up (FU) examination comprised International Knee Documentation Committee (IKDC) 2000, Laxitester (ORTEMA Sport Protection, Markgroeningen, Germany) measurement, and radiograph evaluation. Power calculation was performed to achieve a 95% confidence interval and 80% power on the base of 7-point IKDC subjective difference between the groups. RESULTS: A total of 53 patients (83% FU) were examined at 63.2 ± 4.7 months after surgery: 28 patients in the DB group and 25 patients in the SB group. IKDC subjective (SB: 92.8 ± 6.2, DB: 91.6 ± 7.1; P = .55) and objective scores (grade A SB/DB 20%/25%, B SB/DB 72%/57%, C SB/DB 8%/18%, D SB/DB 4%/0%; P = .45) showed no differences comparing both groups. The Laxitester measurements showed no significant difference in regard to anterior-posterior translation in neutral, internal, and external rotation or to rotation angles (P = .79). No difference was seen between the groups regarding osteoarthritic changes and tunnel widening. CONCLUSIONS: At the 5-year FU, no advantage for either the DB or SB technique in ACL-R can be seen with regard to patient-related and objective outcome measures. LEVEL OF EVIDENCE: Level I, prospective randomized controlled clinical trial.