Histology of tendon and enthesis - suitable techniques for specific research questions.

The musculoskeletal system consists of different components comprising a wide range of tissue types, with tendons being one part. Tendon degeneration or rupture have a high prevalence in all age groups, often with poor outcomes of surgical treatment such as chronic pain and high re-tear rates. Therefore, much effort has been directed to further develop diagnostic and therapeutic methods as well as reconstruction techniques, including using adequate placeholders or implants. Diagnostic approaches and advanced stages of preclinical studies will inevitably include histological examination of the pathologically affected tissue. The present study presents adequate tendon-related, histological techniques, including the embedding of soft- and hard-tissue samples in different media. Consideration is also given to samples containing residual implant materials or having been subjected to standard staining protocols and immunohistochemical procedures. The study further examines cells and tendon structure to detect degenerative, fibrotic or inflammatory conditions and possible foreign-body responses to implanted materials. Infraspinatus tendons from preclinical studies carried on rat and sheep samples, as well as human biceps tendon samples, have been used as example materials.

Do biomarkers allow a differentiation between osteonecrosis of the femoral head and osteoarthritis of the hip? - a biochemical, histological and gene expression analysis.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a devastating disease of the hip joint. Its early diagnosis is crucial to increase the chances of joint preserving, yet difficult due to similarities with osteoarthritis (OA) of the hip in its clinical appearance. The purpose of this study was to enhance the understanding of ONFH and its pathologic processes in contrast to OA and to identify serum biomarkers helping to improve the diagnosis of the disease. DESIGN: Bone and bone marrow samples were collected from 24 patients diagnosed with OA and 25 patients with ONFH during total hip replacement surgery. RNA was isolated, histological examination, determination of free reactive oxygen species as well as gene expression and biomarker analysis were performed. RESULTS: Histological analysis revealed differences in the structural and cellular pattern between the groups. Gene expression analysis revealed a significant upregulation for the genes ASPN, COL1A1, COL2A1 and IL6 and a significant downregulation for HIF1A in ONFH compared to OA group. Analysis of serum biomarkers showed significant differences between the groups for asporin and adiponectin. A final logistical regression model including the parameters adiponectin, asporin and HIF 1α was overall significant, explained 34.5 % of variance and classified 74.5 % of the cases correctly. CONCLUSION: The combination of adiponectin, asporin and HIF 1α as serum biomarkers revealed a classification accuracy of 74.5 %. The information provided in this study may help to enhance the understanding of pathologic processes in ONFH and to elaborate further aspects of prediction and treatment.

Small-sized magnesium cylinders influence subchondral bone quality in osteoarthritic rabbits - an in vivo pilot study.

No optimal therapy exists to stop or cure chondral degeneration in osteoarthritis (OA). While the pathogenesis is unclear, there is consensus on the etiological involvement of both articular cartilage and subchondral bone. Compared to original bone, the substance of sclerotic bone is mechanically less solid. The osteoproliferative effect of Mg has been shown repeatedly during development of Mg-based osteosynthesis implants. The aim of the present study was to examine the influence of implanted high-purity Mg cylinders on subchondral bone quality in a rabbit OA model. 10 New Zealand White rabbits received into the knee either 20 empty drill holes or 20 drill holes, which were additionally filled with one Mg cylinder each. Follow-up was at 8 weeks. Micro-computed tomography (µCT) was performed. After euthanasia, cartilage condition was determined, bone samples were collected and processed for histological evaluation and elemental imaging by micro-X-ray fluorescence spectrometry (µXRF). Articular cartilage collected post-mortem showed different stages of lesions, from mild alterations up to exposed subchondral bone, which tended to be slightly lower in animals with implanted Mg cylinders. µCT showed significantly increased bone volume in the Mg group. Also, histological evaluation revealed distinct differences. While right, operated limbs did not show any significant difference, left, non-operated controls showed significantly less changes in articular cartilage in the Mg group. A distinct influence of implanted cylinders of pure Mg on subchondral bone of osteoarthritic rabbits was shown. Subsequent evaluations, including other time points and alternative alloys, will show if this could alter OA progression.

MgNd2 alloy in contact with nasal mucosa: an in vivo and in vitro approach.

Biodegradable and biocompatible magnesium alloys appear to be very promising not only for temporary clinical application but also for developing deformable and degradable medical implants. This study analyzes the in vivo degradation behavior and the impact on the paranasal sinuses of the highly ductile Mg-2 wt%Nd alloy (MgNd2) in order to provide a basis for a satisfying stent system for the therapy of a chronic sinusitis. Moreover, in vitro tests were carried out on primary porcine nasal epithelial cells (PNEC). For the in vivo tests, cylindrical MgNd2 specimens were implanted into the sinus' mucosa of minipigs. During and after a total period of 180 days the long-term biodegradation and biocompatibility properties after direct contact with the physiological tissue were analyzed. Biodegradation was investigated by measuring the mass and volume losses of the MgNd2 specimens as well as by performing element analyses to obtain information about the degradation layer. The influence on the surrounding tissue of paranasal sinuses was evaluated by endoscopic and histopathological examinations of the mucosa. Here, only a locally unspecific chronic infection was found. The degradation rate showed a maximum after 45 days postsurgery and was determined to decrease subsequently. In vitro experiments using PNEC showed adequate biocompatibility of MgNd2. This study demonstrates a good in vivo biocompatibility for MgNd2 in the system of paranasal sinuses and underlines the promising properties of alloy MgNd2 for biodegradable nasal stent applications.

A novel biodegradable frontal sinus stent (MgNd2): a long-term animal study.

The frontal sinus recess consists of anatomically narrow passages that are prone to stenosis in endonasal frontal sinus surgery for chronic sinus disease. Over the past 100 years, diverse frontal sinus stents have been developed and evaluated in clinical and animal studies. However, superinfection, formation of granulations tissue, stent dislocation and late stenosis of the duct have remained challenges and subject of debate in the literature. Currently developed biodegradable materials, including rare earth-containing magnesium alloys are promising candidates for application as temporary implant materials. The Mg 2 % wt Nd alloy (MgNd2) was used to design a nasal stent that fit the porcine anatomy. In the current study, we evaluate biocompatibility, biodegradation and functionality of a frontal sinus stent in 16 minipigs over 6 months. Intraoperative endoscopy revealed free stent lumen in all cases. Blood examination and clinical examinations indicated no systematic or local inflammation signs. The histopathology and elements analysis showed a very good biocompatibility. The µ-computed tomography-based volumetric analysis showed substantial stent degradation within 6 months. Our MgNd2 based stent appears to be a promising, solid basis for the development of a frontal sinus stent for clinical use.

Laser-structured spike surface shows great bone integrative properties despite infection in vivo.

Implant associated infections can result in devastating consequences for patients. One major cause is the formation of bacterial biofilms, which result in increased resistance against antimicrobial therapeutics. A reduction of implant associated infections can be achieved by functionalization of implant surfaces. The generation of three dimensional surface structures by femtosecond laser ablation is one method to fabricate bacterial repellent large scaled surfaces without altering the material chemical composition. The challenge is to reduce bacterial growth while improving cellular ongrowth. For this purpose, spike structures were created as small as possible by used fabrication method on cubic Ti90/Al6/V4-rods and their effectiveness against bacterial colonization was compared to unstructured Ti90/Al6/V4-rods. Rods were implanted in the rat tibia and infected intraoperatively with 103 CFU of Staphylococcus aureus. Besides clinical behaviour and lameness, the vital bacterial biomass, morphological appearance and the volume of eukaryotic cells were determined on the implant surface after 21 days. Bone alterations were examined by radiological and histological techniques. Unexpectedly, the laser-structured implants did not show a lower bacterial load on the implant surface and less severe infection related bone and tissue alterations compared to the group without structuring. Simultaneously, a better bony integration and a higher cellular colonization with eukaryotic cells was detected on the laser-structured implants. These findings don't support the previous in vitro results. Nevertheless, the strong integration into the bone is a powerful argument for further surface modifications focussing on the improvement of the antibacterial effect. Additionally, our results underline the need for in vivo testing of new materials prior to clinical use.

Biocompatibility and degradation of LAE442-based magnesium alloys after implantation of up to 3.5years in a rabbit model.

UNLABELLED: Magnesium as basic implant material has long been the center of orthopedic research. Latest progress is achieved with a European certification and clinical use of a magnesium based compression screw. However, long term studies with implantation duration that exceed one year considerably do not exist. The present examinations analyzed the degradation progress from nine months to 3.5year after implantation of cylindrical pins into the medullary cavity of New Zealand White rabbits. Evaluation included clinical assessment, in vivo µ-computed tomography, analysis of the implants by three-point-bending and examination of the adjacent tissue by means of histology and of inner organs by mass- and optical emission spectrometry using inductively coupled plasma. Clinical acceptance was without objections in all animals. Immoderate reaction of the surrounding bone could be found in neither of the applied techniques. While in vivo µ-computed tomography showed a very slow degradation rate up to 72weeks, three-point-bending revealed a percentage loss of F(max) of 41.1% for implants after 9months implantation and 88.47% for the implant after 3.5years implantation. Although the total amounts of RE detected in the inner organs were very low, the organs of rabbits with LAE442 cylinders showed 10-20-fold increased concentrations of the alloying elements lanthanum, cerium, neodymium and praseodymium compared to animals without any implanted material. STATEMENT OF SIGNIFICANCE: This is the first animal study investigating the degradation process of a magnesium alloy in vivo for up to 3.5years. Currently available data from different other in vivo studies cover only implantation durations up to one year. Therefore, the analysis of these long-time effects in the present study is highly significant and of great interest. Comprehensive outcome achieved by different techniques was assessed. The degradation process was slow and homogenous. Maximum applied force (F(max)) reduced by 41.1% for implants after 9months and by 88.47% for the implant after 3.5years implantation. Total amounts of RE detected in the inner organs were very low; the organs of rabbits with LAE442 cylinders showed 10-20-fold increased concentrations.

In vitro and in vivo corrosion of the novel magnesium alloy Mg-La-Nd-Zr: influence of the measurement technique and in vivo implant location.

For the evaluation of new magnesium-based alloys, many different in vitro and in vivo methods are used. It was the aim of the current study to perform in vitro and in vivo corrosion studies of the new alloy Mg-La-Nd-Zr for its evaluation as a promising new degradable material and to compare commonly used evaluation methods. Die casted and subsequent extruded cylindrical pins (Ø1.5 mm; length 7 mm, [Formula: see text]) were implanted subcutaneously ([Formula: see text]), intramuscular ([Formula: see text]) and intramedullary ([Formula: see text]) in female Lewis rats with a postoperative follow up of 8 weeks; subsequent µ-computed tomographical analyses (XTremeCT and µCT80) were performed as well as weight analysis prior to and after implantation. Cubes (5 mm × 4 mm × 4 mm; surface area, 1.12 cm(2); [Formula: see text]) were used for in vitro corrosion (HBSS and RPMI 1640 + 10% FBS medium) and cytocompatibility studies (L929 cells). First of all it could be stated that implant location strongly influences the in vivo corrosion rate. In particular, intramedullary implanted pins corroded faster than pins in a subcutaneous or intramuscular environment. Considering the different evaluation methods, the calculated ex vivo µCT-based corrosion rates resulted in comparable values to the corrosion rates calculated by the weight loss method, especially after chromatic acid treatment of the explanted pins. The in vitro methods used tend to show similar corrosion rates compared to in vivo corrosion, especially when a RPMI medium was used, and therefore are suitable to predict corrosion trends prior to in vivo studies. Regarding cytocompatibility, the novel magnesium alloy Mg-La-Nd-Zr showed sufficient cell viability and therefore can be considered as a promising alloy for further applications.

Magnesium-based bone implants: immunohistochemical analysis of peri-implant osteogenesis by evaluation of osteopontin and osteocalcin expression.

The functions of some bone proteins, as osteopontin (OPN) and osteocalcin (OC), have been discovered by the latest studies. This fact suggests the possibility of their immunodetection to characterize peri-implant osteogenesis and implant impact on it. Cylindrical pins of Mg alloys (MgCa0.8, LAE442, ZEK100, LANd442) and titanium alloy (TiAl6V4) were implanted into the tibial medullae of 46 rabbits. Each group was divided regarding to implant duration (3 and 6 months). Bone samples adjacent to the implants were decalcified and treated with routine histological and immunohistochemical protocols using OC and OPN-antibodies. OC was detected in matrix of compact bone, but very rarely in osteoid and bone cells. OPN was detected intracellularly and in osteoid. After 3 months, the highest level of both markers was found in titanium group, followed by LAE442-group. In contrast to LAE442 and TiAl6V4, the other Mg alloys showed increasing levels of OC after 6 months. Lower levels of OP and OC compared to the control group are related to the continuous implant degradation and instability of bone-implant interface in early post-surgical period. Reduced marker's expression in LAE442 and TiAl6V4 groups after 6 months may indicate stabilization of bone-implant interface and completion of peri-implant neo-osteogenesis. Declining characters of OC and OPN expression over the implantation time, as well as their lowest levels in late post-surgical term, suggest a more appropriate biocompatibility of LAE442, which therefore seems to be the most preferable of the tested materials for the use in orthopaedic applications.