Introduction: Various antibiotic coatings have been proposed to prevent bacteria colonization and infection of orthopaedic implants. While most of the available technologies seem to provide an effective implant protection from infection, unknown long-term effects of antibiotic coatings raise some concerns for extensive application. Aim of the present study was to develop and test a new fast-resorbable antibacterial carrier to be used as a temporary coating to prevent early bacteria colonization of metallic implants.

Methods: The patented tested hydrogel is a co-polimer comprising hyaluronic acid (HA) and a biocompatible polyester (poly-lactic acid) with or without polyethylene glycol chains to further modulate hydrophilicity and anti-fouling characteristics of the compound. The HA derivative is then added to water and mixed, just before its use, with the chosen antibacterial agent. For the purpose of this study, different HA-PLA derivatives have been tested, with two vancomycin and tobramycin concentrations and manually spread to uniformly cover the surface of a titanium specimen. To evaluate the release of vancomycin or tobramycin, high performance chromatographic analysis (HPLC) was carried out.

Results: Antibacterial hydrogels provided vancomycin release ranging from 47 % to 80 % in two hours to 100 % (complete release) in 24 to 72 hours, with antibiotic concentrations up to 400 times the minimum inhibiting concentration. The combined release of the two antibiotics (1 % w/v) showed 26.8 % release of vancomycin and 35.8 % of tobramycin at 2 hours and complete release at 72 hours. Doubling antibiotic concentration (2 % w/v), yielded 56.6 % and 76.6 % antibiotic release, respectively for vancomycin and tobramycin at 2 hours and complete release at 48 hours.

Discussion and Conclusion: HA chemical derivatization with polyesters leads to the formation of copolymers which can be used to produce antibacterial hydrogels with promising applications in the orthopedic field. These antibacterial hydrogels are in fact easily prepared and spread over a surface, showing the ability of releasing high concentrations of antibiotics for a desired, limited, period of time. Adding antibiotics to the hydrogel just before its use, allows customized antibiotic choice and dosing, avoiding shelf-life problems.

Aim: This study wants to investigate whether the administration of stromal stem cells (SSC) in a platelet-rich plasma (PRP) scaffold could promote angiogenesis which resulted in a better allograft integration.

Methods: surgery: A monolateral resection of 3cm segment of the metatarsus, was perfomed in 10 adult cross-breed sheep (3–4 years old), weighting 60–70 kg.

Isolation and ex-vivo expansion of SSC: nucleated cells were isolated with density gradient and expanded ex-vivo with alpha-MEM containing 20% FCS.

Radiographic and histomorphometric analysis: Radiographs were made after surgery and after 1, 2 and 4 months. Histomorphometric studies were carried out to study the defect and the new bone formation at the implant site

Results: Union had occurred in all the 5 animals of the SSC group after 4 months as observed radiographically and morphologically, while in the control group the osteotomy line was still visible. Histomorphometric analysis demonstrated a higher % of new-bone formation in both the host (%section quadrant) and the grafted bone in SSC animals.

Conclusions: Results presented suggest that SSC in PRP-based scaffold have improved allograft integration. In conclusion the application of this surgical approach may result in an increased and accelerated bone graft integration, reducing the time required for bone healing and increasing the chances of a successful bone implant.

Aims:. To test the effect of different surface roughness and fluorohydroxyapatite (FHA) coating on osteoblast-like cell (MG63) viability, proliferation, differentiation and synthetic activity, then to compare the various surfaces tested and try to identify an osteoblast parameter that can better explain the different behaviour of the tested surfaces observed in previous in vivo studies.

Methods: The tested materials were made of Ti6Al4V coated with Ti and with Ti plus FHA with different roughness; they can be divided into four groups: low roughness (LR; Ra: 5.9 B5m), low roughness plus FHA coating (LR+FHA; Ra: 5.6 B5m), high roughness (HR; Ra: 22.5 B5m), high roughness plus FHA coating (HR+FHA; Ra: 21.2 B5m). MG63 were cultivated on 6 samples of each group and on polystyrene as control; after 72 hours the proliferation assay (WST-1) was done, alkaline phosphatase activity (ALP) was determined and the synthesis of osteocalcin (OC), type 1 collagen (CICP), transforming growth factor α 1 (TGF-A71), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-a) were measured. Samples of each material were randomly processed for analysis with a scanning electron microscope (SEM).

Results: Cells proliferated on biomaterials more slowly than in the control group (p < 0.0001), the proliferation rate was higher on FHA-coated LR than uncoated HR (p = 0.037). CICP production was positively affected by the LR surface (p = 0.001) as compared to controls, while it was significantly lower (p = 0.0001) in the HR surfaces. Compared to controls, LR and HR surfaces led to enhanced production of TGF-A71, further improved by FHA (FHA-coated LR: p = 0.007; FHA-coated HR p < 0.0001 respectively). ALP, OC, IL-6 levels were not significantly different from the controls

Conclusions: Results suggest that CICP production could be useful in predicting the in vivo osteointegration rate of biocompatible biomaterials observed in previous studies.

When investigating orthopaedic biomaterials and tissue engineered devices, biological investigations by means of in vitro and in vivo tests are mandatory to obtain a overall picture of biocompatibility and therapeutic efficacy. However, various aspects requiring careful consideration should be kept in mind and can explain the complex situations encountered by researchers when the skeletal tissue is involved. This presentation aimed to summarize some useful information in improving in vivo methodology to test present and future therapies for orthopaedic surgery. Some in vivo biological tests to study innovative reconstructive surgical techniques are summarized on the basis of the experience of the Experimental Surgery Department –IOR.

After in vitro and in vivo biocompatibility tests, for the study of bone defect healing and of biomaterial osteo-inductive properties the subcutaneous and intramuscular implants are usually performed in laboratory animals while osteoconduction and bone healing evaluation require the development of “nonunions” (sites that never achieve functional bone continuity) and “critical size defects” (the smallest defect that will heal with less than 10% bony growth) models. Biomaterial osteointegration properties are investigated by means of metaphyseal, diaphyseal and intramedullary implantation. The use of pathological animals is also recommended to take into account the clinical situation where biomaterials are often implanted in aged and osteoporotic patients. As far as articular cartilage pathology is concerned, chondral and osteochondral “critical size defects” may be performed and the development of osteoarthritic animals could be also useful.

At different experimental times post-explantation evaluations by means of radiology, histology, histomorphometry and biomechanics provide a complete characterization of biomaterials and biotechnologies showing their potential therapeutic efficacy for skeletal reconstruction.

In vivo studies provide important pre-clinical information on new biomaterials and biotechnologies for the skeletal reconstruction Among the factors that are increasingly improving the reliability of in vivo testing are the continuous improvement in knowledge on bone biology and comparative science between humans and animals, the awareness that animal suffering should be reduced as much as possible, and, finally, the amount and the accuracy of in vivo post-explantation findings.