Physiological cell bioprinting density in human bone-derived cell-laden scaffolds enhances matrix mineralization rate and stiffness under dynamic loading.

Human organotypic bone models are an emerging technology that replicate bone physiology and mechanobiology for comprehensive in vitro experimentation over prolonged periods of time. Recently, we introduced a mineralized bone model based on 3D bioprinted cell-laden alginate-gelatin-graphene oxide hydrogels cultured under dynamic loading using commercially available human mesenchymal stem cells. In the present study, we created cell-laden scaffolds from primary human osteoblasts isolated from surgical waste material and investigated the effects of a previously reported optimal cell printing density (5 × 106 cells/mL bioink) vs. a higher physiological cell density (10 × 106 cells/mL bioink). We studied mineral formation, scaffold stiffness, and cell morphology over a 10-week period to determine culture conditions for primary human bone cells in this microenvironment. For analysis, the human bone-derived cell-laden scaffolds underwent multiscale assessment at specific timepoints. High cell viability was observed in both groups after bioprinting (>90%) and after 2 weeks of daily mechanical loading (>85%). Bioprinting at a higher cell density resulted in faster mineral formation rates, higher mineral densities and remarkably a 10-fold increase in stiffness compared to a modest 2-fold increase in the lower printing density group. In addition, physiological cell bioprinting densities positively impacted cell spreading and formation of dendritic interconnections. We conclude that our methodology of processing patient-specific human bone cells, subsequent biofabrication and dynamic culturing reliably affords mineralized cell-laden scaffolds. In the future, in vitro systems based on patient-derived cells could be applied to study the individual phenotype of bone disorders such as osteogenesis imperfecta and aid clinical decision making.

Impact of degeneration and material pairings on cartilage friction: Cartilage versus glass.

The association of knee joint osteoarthritis and altered frictional properties of the degenerated cartilage remains ambiguous, because previous in vitro studies did not consider the characteristic loads and velocities during gait. Therefore, the aim of this study was to quantify the friction behavior of degenerated human cartilage under characteristic stance and swing phase conditions. A dynamic pin-on-plate tribometer was used to test the tribological systems of cartilage against cartilage and cartilage against glass, both with synthetic synovial fluid as lubricant. Using the International Cartilage Repair Society classification, the cartilage samples were assigned to a mildly or a severely degenerated group before testing. Friction coefficients were calculated under stance and swing phase conditions at the beginning of the test and after 600 s of testing. The most important finding of this study is that cartilage against glass couplings displayed significantly higher friction for the severely degenerated samples compared to the mildly degenerated ones, whereas cartilage against cartilage couplings only indicated slight tendencies under the observed test conditions. Consequently, care should be taken when transferring in vitro findings from cartilage against cartilage couplings to predict the friction behavior in vivo. Therefore, we recommend in vitro tribological testing methods which account for gait-like loading conditions and to replicate physiological material pairings, particularly in preclinical medical device validation studies.

A Bioinspired Orthopedic Biomaterial with Tunable Mechanical Properties Based on Sintered Titanium Fibers.

Inadequate mechanical compliance of orthopedic implants can result in excessive strain of the bone interface, and ultimately, aseptic loosening. It is hypothesized that a fiber-based biometal with adjustable anisotropic mechanical properties can reduce interface strain, facilitate continuous remodeling, and improve implant survival under complex loads. The biometal is based on strategically layered sintered titanium fibers. Six different topologies are manufactured. Specimens are tested under compression in three orthogonal axes under 3-point bending and torsion until failure. Biocompatibility testing involves murine osteoblasts. Osseointegration is investigated by micro-computed tomography and histomorphometry after implantation in a metaphyseal trepanation model in sheep. The material demonstrates compressive yield strengths of up to 50 MPa and anisotropy correlating closely with fiber layout. Samples with 75% porosity are both stronger and stiffer than those with 85% porosity. The highest bending modulus is found in samples with parallel fiber orientation, while the highest shear modulus is found in cross-ply layouts. Cell metabolism and morphology indicate uncompromised biocompatibility. Implants demonstrate robust circumferential osseointegration in vivo after 8 weeks. The biometal introduced in this study demonstrates anisotropic mechanical properties similar to bone, and excellent osteoconductivity and feasibility as an orthopedic implant material.

Pediatric aseptic lower leg fracture nonunion.

PURPOSE: Lower leg nonunion in pediatric patients is a rarity. Therefore, eight European pediatric trauma units retrospectively analyzed all patients younger than 18 years suffering lower leg fractures resulting in aseptic nonunion. METHODS: Thirteen children and adolescents less than 18 years old (2 girls and 11 boys) diagnosed with aseptic nonunion of the tibia and/or fibula were evaluated. In all patients, epidemiological data, mechanism of injury, fracture configuration, and the initial treatment concept were assessed, and the entire medical case documentation was observed. Furthermore, potential causes of nonunion development were evaluated. RESULTS: The mean age of patients was 12.3 years with the youngest patient being seven and the oldest being 17 years old. Open fractures were found in six out of thirteen patients (46%). Nonunion was hypertrophic in ten and oligotrophic in three patients. Mean range of time to nonunion occurrence was 7.3 ± 4.6 months. Nonunion healing resulting in complete metal removal was found in 12 out of 13 patients (92%), only in one case of a misinterpreted CPT type II osseous consolidation could not be found during the observation period. Mean range of time between surgical nonunion revision and osseous healing was 7.3 months as well. CONCLUSION: If treatment principles of the growing skeleton are followed consistently, aseptic nonunion of the lower leg remains a rare complication in children and adolescents. Factors influencing the risk of fracture nonunion development include patient's age, extended soft tissue damage, relevant bone loss, and inadequate initial treatment.

The Effect of Polymethyl Methacrylate Augmentation on the Primary Stability of Cannulated Bone Screws in an Anterolateral Plate in Osteoporotic Vertebrae: A Human Cadaver Study.

Study Design Cohort study. Objective Expandable anterolateral plates facilitate the reduction of posttraumatic deformities of thoracolumbar spine injuries and are commonly used in cases of unstable injuries or compromised bone quality. In this in vitro study, the craniocaudal yield load of the osseous fixation of an anterior angular stable plate fixation system and the effect of polymethyl methacrylate (PMMA) screw augmentation on the primary stability of the screw-bone interface during kyphosis reduction was evaluated in 12 osteoporotic human thoracolumbar vertebrae. Methods The anterolateral stabilization device used for this study is comprised of two swiveling flanges and an expandable midsection. It facilitates the controlled reduction of kyphotic deformities in situ with a geared distractor. Single flanges were attached to 12 thoracolumbar vertebrae. Six specimens were augmented with PMMA by means of cannulated bone screws. The constructs were subjected to static, displacement-controlled craniocaudal loading to failure in a servohydraulic testing machine. Results The uncemented screws cut out at a mean 393 ± 66 N, whereas the cemented screws showed significantly higher yield load of 966 ± 166 N (p < 0.02). We detected no significant correlation between bone mineral density and yield load in this setting. Conclusion Our results indicate that PMMA augmentation is an effective method to increase two- to threefold the primary stability of the screw-bone interface of an anterolateral spine stabilization system in osteoporotic bone. We recommend it in cases of severely compromised bone quality to reduce the risk of screw loosening during initial kyphosis correction and to increase long-term construct stability.

Outcome after severe multiple trauma: a retrospective analysis.

BACKGROUND: Aim of this study was to evaluate prognosis of severely injured patients. METHODS: All severely injured patients with an Injury Severity Score (ISS) ≥ 50 were identified in a 6-year-period between 2000 and 2005 in German Level 1 Trauma Center Murnau. Data was evaluated from German Trauma Registry and Polytrauma Outcome Chart of the German Society for Trauma Surgery and a personal interview to assess working ability and disability and are presented as average. RESULTS: 88 out of 1435 evaluated patients after severe polytrauma demonstrated an ISS ≥ 50 (6.5%), among them 23% women and 77% men. 66 patients (75%) had an ISS of 50-60, 14 (16%) 61-70, and 8 (9%) ≥ 70. In 27% of patients trauma was caused by motor bike accidents. 3.6 body regions were involved. Patients had to be operated 5.3 times and were treated 23 days in the ICU and stayed 73 days in hospital. Mortality rate was 36% and rate of multi-organ failure 28%. 15% of patients demonstrated severe senso-motoric dysfunction as well as residues of severe head injury. 25% recovered well or at least moderately. 29 out of 56 survivors answered the POLO-chart. A personal interview was performed with 13 patients. The state of health was at least moderate in 72% of patients. In 48% interpersonal problems and in 41% severe pain was observed. In 57% of patients problems with working ability regarding duration, as well as quantitative and qualitative performance were observed. Symptoms of post-traumatic stress disorder were found in 41%. The more distal the lesions were located (foot/ankle) the more functional disability affected daily life. In only 15%, working ability was not impaired. 8 out of 13 interviewed patients demonstrated complete work disability. CONCLUSIONS: Even severely injured patients after multiple trauma have a good prognosis. The ISS is an established tool to assess severity and prognosis of trauma, whereas prediction of clinical outcome cannot be deducted from this score.

The removal of Al2O3 particles from grit-blasted titanium implant surfaces: effects on biocompatibility, osseointegration and interface strength in vivo.

For the improvement of surface roughness and mechanical interlocking with bone, titanium prostheses are grit-blasted with Al(2)O(3) particles during manufacturing. Dislocated Al(2)O(3) particles are a leading cause of third-body abrasive wear in the articulation of endoprosthetic implants, resulting in inflammation, pain and ultimately aseptic loosening and implant failure. In the present study, a new treatment for the removal of residual Al(2)O(3) particles from grit-blasted, cementless titanium endoprosthetic devices was investigated in a rabbit model. The cleansing process reduces residual Al(2)O(3) particles on titanium surfaces by up to 96%. The biocompatibility of the implants secondary to treatment was examined histologically, the bone-implant contact area was quantified histomorphometrically, and interface strength was evaluated with a biomechanical push-out test. Conventional grit-blasted implants served as control. In histological and SEM analysis, the Al(2)O(3)-free implant surfaces demonstrated uncompromised biocompatibility. Histomorphometrically, Al(2)O(3)-free implants exhibited a significantly increased bone-implant contact area (p=0.016) over conventional implants between both evaluation points. In push-out testing, treated Al(2)O(3)-free implants yielded less shear resistance than conventional implants at both evaluation points (p=0.018). In conclusion, the new surface treatment effectively removes Al(2)O(3) from implant surfaces. The treated implants demonstrated uncompromised biocompatibility and bone apposition in vivo. Clinically, Al(2)O(3)-free titanium prostheses could lead to less mechanical wear of the articulating surfaces and ultimately result in less aseptic loosening and longer implant life.

Vertebroplasty with high-viscosity polymethylmethacrylate cement facilitates vertebral body restoration in vitro.

STUDY DESIGN: In vitro biomechanical study on 6 fresh frozen human thoracolumbar spine specimens. OBJECTIVE: Using a novel high viscosity polymethylmethacrylate (PMMA) cement and vertebroplasty kit to correct the kyphosis angle of wedge compression fractures (AO/ASIF 1.2). SUMMARY OF BACKGROUND DATA: Vertebroplasty is typically used to stabilize vertebral compression fractures in situ without correcting kyphosis, with the main target to reduce pain and disability. The vertebroplasty system investigated in this study comprises a high viscosity PMMA cement and uses a hydrostatic pressure hand piece for enhanced cement allocation and flow control. A recent clinical trial demonstrated a significantly reduced incidence of cement leakage with this system. METHODS: Six spinal segments (Th11-L1 and Th12-L2) were loaded in a spine tester with pure moments of 7.5 Nm in lateral bending, flexion/extension and axial rotation. The segmental range of motion (ROM) was continuously recorded. The tested states of the specimens were: intact (a), fractured (b), treated with vertebroplasty (c), after loading with 50 to 250 N (d), 50 to 450 N (e) and 50 to 650 N (f) of 1000 cycles each. In each state (a-f), the kyphosis angle was documented fluoroscopically. RESULTS: Kyphosis angle was significantly reduced between intact and fractured states (P<0.02). Between treated and fractured states, we found highly significant difference (P<0.001), indicating full correction. During 3000 loading cycles (50-250, 50-450, and 50-650 N), the kyphosis angle remained constant compared to the treated state (P=1.0). We noted a logistic relationship between injected cement volume and extent of kyphosis correction (R=0.89, P<0.001). In the fractured state, the ROM in flexion/extension increased to 252% of the intact state (P<0.001). The vertebroplasty treatment decreased ROM to 72% of fractured state in flexion/extension (P<0.001). Macroscopic inspection of the vertebrae after testing showed an intact interface and tight mechanical interlocking of cement filling and trabecular bone. CONCLUSION: High viscosity vertebroplasty effectively reduced and stabilized thoracolumbar wedge compression fractures and may represent a one-step solution for restoring vertebral body dimensions following thoracolumbar compression fractures, while minimizing the risk of cement leakage and associated complications in vivo.

The effect of surface modification of a porous TiO2/perlite composite on the ingrowth of bone tissue in vivo.

The porous TiO2/perlite composite Ecopore is a synthetic biomaterial with possible clinical application in bone substitution. In our previous work, we demonstrated that surface modification of Ecopore with fibronectin (FN) enhanced spreading and growth of human osteoblasts in vitro. In the present study, we implanted untreated, alkaline-etched and FN-coated Ecopore cylinders into critical size defects of rabbit femora and applied pulsed polychrome sequence staining. After 6 weeks, sections of the implants were investigated via conventional and fluorescence microscopy. A partial ingrowth of bone matrix into the pore system of the Ecopore implants was observed. At the contact zones, the bone appeared to be directly connected to the implant without detectable gaps. Defect healing was complete within 6 weeks, while fibrous tissue generation or inflammation were absent in the implant modification groups, demonstrating basic Ecopore biocompatibility. The mean bone apposition rates within the implant cross-section were 4.1+/-0.6 microm/day (p<0.001) in the FN-coated group and 3.3+/-0.5 microm/day (p<0.05) in the NaOH-etched group. In both treated Ecopore modification groups, the apposition rates were significantly higher than in the non-modified control (2.9+/-0.6 microm/day), indicating bone growth stimulation by pre-treatment. Energy-dispersive X-ray analysis confirmed that significantly more bone tissue was formed inside the pores of the FN-coated implants compared to the unmodified control. The cross-sectional areas identified as ingrown bone amounted to 18.5+/-6.1% (p<0.05) in the FN group, 13.4+/-5.1% (p>0.05) in the NaOH-etched group and 10.2+/-5.5% in the unmodified group. In summary, we conclude that bone tissue tolerates Ecopore well and that tissue ingrowth can be enhanced by etching and coating with FN.

In vitro behavior of a porous TiO2/perlite composite and its surface modification with fibronectin.

In this study, we introduce a porous composite material, termed "Ecopore", and describe in vitro investigation of the material and its modification with fibronectin. The material is a sintered compound of rutile TiO2 and the volcanic silicate perlite with a macrostructure of interconnecting pores. It is both inexpensive and easy to manufacture. We first investigated Ecopore for corrosion and leaching of elements in physiological saline. The corrosion supernatants did not contain critical concentrations of toxic trace elements. In an in vitro model, human primary osteoblasts (HOB) were cultured directly on Ecopore. HOB grew on the composite as well as on samples of its single constituents, TiO2 and perlite glass, and remained vital, but cellular spreading was less than on tissue culture plastic. The pro-inflammatory cytokines IL-1 and TNF-alpha were below detection limits in HOB culture supernatants, whereas IL-6 was detectable on a low level. To enhance cellular attachment and growth, the surface of the composite was modified by etching, functionalization with aminosilane and coupling of fibronectin. This modification greatly enhanced the spreading of HOB, indicated by vital staining and Sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) metabolism assays. HOB grew on the entire visible surface of porous fibronectin-modified composite, expressing alkaline phosphatase, a mature osteoblast marker. We conclude that Ecopore is non-toxic and sustains HOB growth, cellular spreading being improvable by coating with fibronectin. The composite may be usable in the field of bone substitution.